# Patent

## Title
Hydrogel particles with tunable optical properties and methods for using the same

## Abstract

Hydrogel particles and their use in cytometic applications are described. The hydrogel particles described herein are selectively tunable to have at least one optical property substantially similar to the at least one optical property of a target cell. In this regard, the hydrogel particles provided herein in one aspect, are used as a calibration reagent for the detection of a target cell in a sample.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority from U.S. Provisional Application No. 62/114,004, filed Feb. 9, 2015 and U.S. Provisional Application No. 62/184,192, filed Jun. 24, 2015, the disclosure of each of which is incorporated by reference herein in their entireties.


BACKGROUND OF THE INVENTION
Flow cytometry is a technique that allows for the rapid separation, counting, and characterization of individual cells and is routinely used in clinical and laboratory settings for a variety of applications. The technology relies on directing a beam of light onto a hydrodynamically-focused stream of liquid. A number of detectors are then aimed at the point where the stream passes through the light beam: one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter or SSC). FSC correlates with the cell volume and SSC depends on the inner complexity of the particle (e.g., shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness). As a result of these correlations, different specific cell types exhibit different FSC and SSC, allowing cell types to be distinguished in flow cytometry.
The ability to identify specific cell types, however, relies on proper calibration of the instrument, a process that has relied on the use of purified cells of the cell type of interest. Obtaining these purified cells can require costly, laborious procedures that are prone to batch-to-batch variation. Therefore, there is a need in the art for synthetic compositions with tunable optical properties that can mimic specific cell types in devices such as flow cytometers.
SUMMARY OF THE INVENTION
In one aspect of the invention, a hydrogel particle comprising a polymerized monomer and having at least one surface is provided. The hydrogel particle has at least one optical property that is substantially similar to the at least one optical property of a target cell. The optical property in one embodiment, is a side scatter profile (SSC), forward scatter profile (FSC), a fluorescence emission profile, or a combination thereof. The target cell can be any target cell that the user specifies. For example, in one embodiment, the target cell is an immune cell, stem cell or cancer cell.
In another aspect, a method for calibrating a cytometric device for analysis of a target cell, is provided. In one embodiment, the method comprises inserting into the device a hydrogel particle having at least one optical property substantially similar to a target cell, wherein the hydrogel particle comprises a polymerized monomer and has at least one surface. The method further comprises measuring the at least one optical property of the hydrogel particle using the cytometric device. The at least one optical property in one embodiment, is used as a reference to detect a target cell in a sample.
In yet another aspect, a method for detecting a target cell in a sample is provided. The method comprises inserting into the device a hydrogel particle having at least one optical property substantially similar to a target cell, wherein the hydrogel particle comprises a polymerized monomer. The method further comprises measuring the at least one optical property of the hydrogel particle using the cytometric device. A sample comprising a plurality of cells is inserted into the cytometric device, and the at least one optical property of individual cells of the plurality are measured. Finally, a determination is made, based on the optical property measurement, whether the target cell or plurality thereof is present in the sample.
In one embodiment of the methods provided herein, the hydrogel particle comprises a biodegradable monomer. In a further embodiment, the biodegradable monomer is a monosaccharide, disaccharide, polysaccharide, peptide, protein, or protein domain. In even a further embodiment, the biodegradable monomer is functionalized with acrylamide or acrylate.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 illustrates the optical properties of disclosed hydrogel particles compared to polystyrene beads.
FIG. 2 depicts the process of producing labeled hydrogel particles of the disclosure.
FIG. 3 provides brightfield and fluorescent images of labeled hydrogel particles of the disclosure.
FIG. 4 illustrates the use of hydrogel particles of the disclosure as calibrants for cell types displaying a variety of optical scattering properties.
FIG. 5 provides dating showing correlation of inter-drop delay for a flow cytometer with hydrogel particle diameter.
FIG. 6 provides brightfield (6A and 6C) and fluorescent (6B and 6D) images of Chinese Hamster Ovary cells (6A and 6B) and hydrogel particles of the disclosure (6C and 6D).
FIG. 7 provides data showing comparison of human buccal cells to hydrogel particles encapsulating different amounts of DNA, as measured by fluorescence-activated cell sorting (FACS).
FIG. 8 provides data for hydrogel particles encapsulating nanoparticles at different concentrations, demonstrating tuning of side scattering independent of forward scattering.
FIG. 9 provides data for hydrogel particles produced with different percentages of polymer, demonstrating tuning of refractive index measured by forward scattering.
FIG. 10 shows one embodiment of hydrogel parameter tuning to match and/or mimic desired cell population metrics.
FIGS. 11 and 12 are diagrams showing embodiments of how to adjust the forward scatter, side scatter and surface properties of a hydrogel particle.
FIG. 13 are scatter plots for various hydrogel particles (A) and (B) and a commercial blood sample (C).



DETAILED DESCRIPTION OF THE INVENTION
The indefinite articles “a” and “an” and the definite article “the” are intended to include both the singular and the plural, unless the context in which they are used clearly indicates otherwise.
“At least one” and “one or more” are used interchangeably to mean that the article may include one or more than one of the listed elements.
Unless otherwise indicated, it is to be understood that all numbers expressing quantities, ratios, and numerical properties of ingredients, reaction conditions, and so forth, used in the specification and claims are contemplated to be able to be modified in all instances by the term “about”.
Several critical calibration measurements for flow cytometers require precise time resolution, such as setting the offset time between lasers, and calculating the delay time between detection and sorting of an object. Due to the fluidic conditions within the instrument, precise setting of these timing parameters requires the use of calibration particles that are the same size as the cells to be analyzed. Timing calibrations are typically performed using polystyrene beads with variable fluorescent intensities to calibrate the response of an excitation source and to set the inter-laser timing delay and sorting delay. Flow cytometers can also be calibrated using forward and side scatter signals which are general measures of size and granularity or complexity of the target sample. These calibrations are crucial for the accurate performance of the cytometer and for any downstream analysis or sorting of cell populations. The disclosed hydrogel particles exhibit tuned scatter properties and are suitable for use as calibration reagents for a range of mammalian or bacterial cell types. Scattering is a standard metric for distinguishing cell types in heterogeneous mixtures for clinical, food safety, and research purposes.
Although polystyrene particles can be used to set inter-laser and sorting delays for some applications, many eukaryotic cell types fall outside of the size range of commercially available polystyrene particles (1-20 μm) making it nearly impossible to accurately calibrate a flow cytometer for these targets. Also, as shown in FIG. 1, polystyrene particles are fundamentally limited in the optical properties that can possess such as side scattering, which is a general measure of cellular complexity. Polystyrene particles are therefore limited in the two most important passive optical measurements used in flow cytometry: FSC (forward scattering), and SSC (side scattering) which measure the size and complexity of the target respectively. Due to these limitations of polystyrene, users must rely on purified cell lines to calibrate fluorescent intensity, inter-laser delay, sort delays, size and cellular complexity for experiments. This is a lengthy and labor-intensive process that increases the cost of flow cytometry validation and research pipelines significantly. More importantly, these calibration cell lines introduce biological variation, causing disparities in the interpretation of data.
Moreover, quality control (QC) for calibration of flow cytometers is also a crucial consideration when these instruments are used for clinical applications, for example, to isolate human T-regulatory cells or stem cells for downstream cellular therapies. The FDA mandates that the sterility, identity, purity, and potency of a cell therapy product be demonstrated before administration to patients (Riley et al. (2009). Immunity 30, pp. 656-665). Contamination of a cellular population with polystyrene QC particles could therefore be problematic, as polystyrene has been implicated in certain cancers. Additionally, a cellular population that is contaminated with a QC standard that is enzymatically degraded or digested internally after administration to a patient potentially overcomes contamination issues, should they arise.
The present invention addresses these and other needs, as discussed below.
In one aspect, a composition comprising a plurality of hydrogel particles is provided, wherein the individual hydrogel particles of the plurality each has one or more optical properties substantially similar to one or more optical properties of a target cell. Each of the individual hydrogel particles of the plurality independently comprises a hydrogel which is synthesized by polymerizing one or more monomers, i.e., to form a homopolymer or copolymer. As discussed further below, the use of bifunctional monomers allows for the further derivatization of hydrogels, e.g., with fluorescent dyes, cell surface markers or epitope binding fragments thereof, or a combination thereof. An example of hydrogel parameter tuning to meet/match desired cell subpopulation metrics is provided at FIG. 10. Methods for tuning the properties of a hydrogel are described herein. The ability to adjust a range of parameters including hydrogel components and concentration of the same allows for the ability to tune a particle to mimic a wide range of cells, for example one of the cell types described herein.
As provided above, in one aspect, the present invention provides individual hydrogel particles each having one or more optical properties substantially similar to one or more optical properties of a target cell. In one embodiment, the one or more optical properties, is a side scatter profile, a forward scatter profile or a secondary marker profile, such as a fluorescence marker profile, for example a fluorescence marker profile of a fluorescently-labeled antibody that binds to the surface of the hydrogel particle. “Substantially similar,” as used herein, denotes at least 40% similar, at least 50% similar, at least 60% similar, at least 70% similar, at least 80% similar, at least 90% similar, at least 95% similar, at least 96% similar, at least 97% similar, at least 98% similar or at least 99% similar.
The present invention is based in part on the unexpected discovery that one or more optical properties of a hydrogel particle can be independently modulated by altering the composition of the hydrogel particle, for example, by altering the amount of initial monomer (or co-monomer) in the composition, by altering the surface functionalization, by altering the amount of a polymerization initiator or by altering the amount of crosslinker. For example, side scattering (SSC) can be modulated without substantially affecting forward scattering (FSC), and vice versa. Furthermore, the optical properties (e.g. refractive index) of hydrogel particles can be tuned without having a substantial effect on density of the particle. This is a surprising and useful feature, as hydrogel particles that serve as surrogates for cells in cytometric methods such as flow cytometry or (fluorescence-activated cell sorting) FACS require a minimal density in order to function in those assays.
In another aspect, a method for producing a hydrogel particle is provided, wherein the hydrogel particle has one or more optical properties substantially similar to the optical properties of one or more target cells. In one embodiment, the hydrogel particle has pre-determined optical properties. The optical property, in one embodiment, is SSC, FSC, fluorescence emission, or a combination thereof.
In yet another aspect, a method of calibrating a cytometric device for analysis of a target cell is provided. In one embodiment, the method comprises (a) inserting into the device a hydrogel particle having optical properties substantially similar to the optical properties of the target cell; b) measuring the optical properties of the hydrogel particle using the cytometric device, thereby calibrating the cytometric device for analysis of the target cell. Cytometric devices are known in the art, and include commercially available devices for performing flow cytometry and FACS.
As provided above, in one aspect of the invention, compositions comprising a plurality of hydrogel particles are provided. A hydrogel is a material comprising a macromolecular three-dimensional network that allows it to swell when in the presence of water, to shrink in the absence of (or by reduction of the amount of) water, but not dissolve in water. The swelling, i.e., the absorption of water, is a consequence of the presence of hydrophilic functional groups attached to or dispersed within the macromolecular network. Crosslinks between adjacent macromolecules result in the aqueous insolubility of these hydrogels. The cross-links may be due to chemical (i.e., covalent) or physical (i.e., VanDer Waal forces, hydrogen-bonding, ionic forces, etc.) bonds. Synthetically prepared hydrogels can be prepared by polymerizing a monomeric material to form a backbone and cross-linking the backbone with a crosslinking agent. As referred to herein, the term “hydrogel” refers to the macromolecular material whether dehydrated or in a hydrated state. A characteristic of a hydrogel that is of particular value is that the material retains the general shape, whether dehydrated or hydrated. Thus, if the hydrogel has an approximately spherical shape in the dehydrated condition, it will be spherical in the hydrated condition.
In one embodiment, a hydrogel particle disclosed herein comprises greater than about 30%, greater than about 40%, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, or greater than about 95% water. In another embodiment, a hydrogel particle has a water content of about 10 percent by weight to about 95 percent by weight, or about 20 percent by weight to about 95 percent by weight, or about 30 percent by weight to about 95 percent by weight, or about 40 percent by weight to about 95 percent by weight, or about 50 percent by weight to about 95 percent by weight, or about 60 percent by weight to about 95 percent by weight, or about 70 percent by weight to about 95 percent by weight, or about 80 percent by weight to about 95 percent by weight.
The hydrogels provided herein, in the form of particles, are synthesized by polymerizing one or more of the monomers provided herein. The synthesis is carried out to form individual hydrogel particles. The monomeric material (monomer) in one embodiment is polymerized to form a homopolymer. However, in another embodiment copolymers of different monomeric units (i.e., co-monomers) are synthesized and used in the methods provided herein. The monomer or co-monomers used in the methods and compositions described herein, in one embodiment, is a bifunctional monomer or includes a bifunctional monomer (where co-monomers are employed). In one embodiment, the hydrogel is synthesized in the presence of a crosslinker. In a further embodiment, embodiment, the hydrogel is synthesized in the presence of a polymerization initiator.
The amount of monomer can be varied by the user of the invention, for example to obtain a particular optical property that is substantially similar to that of a target cell. In one embodiment, the monomeric component(s) (i.e., monomer, co-monomer, bifunctional monomer, or a combination thereof, for example, bis/acrylamide in various crosslinking ratios, allyl amine or other co-monomers which provide chemical functionality for secondary labeling/conjugation or alginate is present at about 10 percent by weight to about 95 percent weight of the hydrogel. In a further embodiment, the monomeric component(s) is present at about 15 percent by weight to about 90 percent weight of the hydrogel, or about 20 percent by weight to about 90 percent weight of the hydrogel.
Examples of various monomers and cross-linking chemistries available for use with the present invention are provided in the Thermo Scientific Crosslinking Technical Handbook entitled “Easy molecular bonding crosslinking technology,” (available at tools.lifetechnologies.com/content/sfs/brochures/1602163-Crosslinking-Reagents-Handbook.pdf, the disclosure of which is incorporated by reference in its entirety for all purposes. For example, hydrazine (e.g., with an NHS ester compound) or EDC coupling reactions (e.g., with a maleimide compound) can be used to construct the hydrogels of the invention.
In one embodiment, a monomer for use with the hydrogels provided herein is lactic acid, glycolic acid, acrylic acid, 1-hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glycerol methacrylate (GMA), glycol methacrylate, ethylene glycol, fumaric acid, a derivatized version thereof, or a combination thereof.
In one embodiment, one or more of the following monomers is used herein to form a hydrogel of the present invention: 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, poly(ethylene glycol) methacrylate, methoxy-poly(ethylene glycol) methacrylate, methacrylic acid, sodium methacrylate, glycerol methacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate or a combination thereof.
In another embodiment, one or more of the following monomers is used herein to form a tunable hydrogel: phenyl acrylate, phenyl methacrylate, benzyl acrylate, benzyl methacrylate, 2-phenylethyl acrylate, 2-phenylethyl methacrylate, 2-phenoxyethyl acrylate, 2-phenoxyethyl methacrylate, phenylthioethyl acrylate, phenylthioethyl methacrylate, 2,4,6-tribromophenyl acrylate, 2,4,6-tribromophenyl methacrylate, pentabromophenyl acrylate, pentabromophenyl methacrylate, pentachlorophenyl acrylate, pentachlorophenyl methacrylate, 2,3-dibromopropyl acrylate, 2,3-dibromopropyl methacrylate, 2-naphthyl acrylate, 2-naphthyl methacrylate, 4-methoxybenzyl acrylate, 4-methoxybenzyl methacrylate, 2-benzyloxyethyl acrylate, 2-benzyloxyethyl methacrylate, 4-chlorophenoxyethyl acrylate, 4-chlorophenoxyethyl methacrylate, 2-phenoxyethoxyethyl acrylate, 2-phenoxyethoxyethyl methacrylate, N-phenyl acrylamide, N-phenyl methacrylamide, N-benzyl acrylamide, N-benzyl methacrylamide, N,N-dibenzyl acrylamide, N,N-dibenzyl methacrylamide, N-diphenylmethyl acrylamide N-(4-methylphenyl)methyl acrylamide, N-1-naphthyl acrylamide, N-4-nitrophenyl acrylamide, N-(2-phenylethyl)acrylamide, N-triphenylmethyl acrylamide, N-(4-hydroxyphenyl)acrylamide, N,N-methylphenyl acrylamide, N,N-phenyl phenylethyl acrylamide, N-diphenylmethyl methacrylamide, N-(4-methyl phenyl)methyl methacrylamide, N-1-naphthyl methacrylamide, N-4-nitrophenyl methacrylamide, N-(2-phenylethyl)methacrylamide, N-triphenylmethyl methacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenyl methacrylamide, N,N′-phenyl phenylethyl methacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine, as described in U.S. Pat. No. 6,657,030, which is incorporated by reference in its entirety herein for all purposes.
Both synthetic monomers and bio-monomers can be used in the hydrogels provided herein, to form synthetic hydrogels, bio-hydrogels, or hybrid hydrogels that comprise a synthetic component and a bio-component (e.g., peptide, protein, monosaccharide, disaccharide, polysaccharide, primary amines sulfhydryls, carbonyls, carbohydrates, carboxylic acids present on a biomolecule). For example, proteins, peptides or carbohydrates can be used as individual monomers to form a hydrogel that includes or does not include a synthetic monomer (or polymer) and in combination with chemically compatible co-monomers and crosslinking chemistries (see for example, the Thermo Scientific Crosslinking Technical Handbook entitled “Easy molecular bonding crosslinking technology,” available at tools.lifetechnologies.com/content/sfs/brochures/1602163-Crosslinking-Reagents-Handbook.pdf, the disclosure of which is incorporated by reference in its entirety for all purposes.). Compatible crosslinking chemistries include, but are not limited to, amines, carboxyls, and other reactive chemical side groups. Representative reactive groups amenable for use in the hydrogels and monomers described herein are provided in Table 1, below.







TABLE 1







Crosslinker reactive groups amenable for bio-monomer conjugation










Target




functional


Reactivity class
group
Reactive chemical group





Amine reactive
—NH2
NHS ester




Imidoester




Penafluorophenyl ester




Hydroxymethyl phosphine


Carboxyl-to-amine reactive
—COOH
Carbodiimide (e.g., EDC)


Sulfhydryl-reactive
—SH
Maeleimide




Haloacetyl (bromo- or




iodo-)




Pyridylisulfide




Thiosulfonate




Vinylsulfonate


Aldehyde-reactive (oxidized
—CHO
Hydrazine


sugars, carbonyls)

Alkoxyamine


Photo-reactive, i.e.,
Random
Diazirine


nonselective, random insertion

Aryl azide


Hydroxyl (nonaqueous)-reactive
—OH
Isocyanate


Azide-reactive
—N3
phosphine









In general, any form of polymerization chemistry/methods commonly known by those skilled in the art, can be employed to form polymers. In some embodiments, polymerization can be catalyzed by ultraviolet light-induced radical formation and reaction progression. In other embodiments, a hydrogel particle of the disclosure is produced by the polymerization of acrylamide or the polymerization of acrylate. For example, the acrylamide in one embodiment is a polymerizable carbohydrate derivatized acrylamide as described in U.S. Pat. No. 6,107,365, the disclosure of which is incorporated by reference in its entirety for all purposes. As described therein and known to those of ordinary skill in the art, specific attachment of acrylamide groups to sugars is readily adapted to a range of monosaccharides and higher order polysaccharides, e.g., synthetic polysaccharides or polysaccharides derived from natural sources, such as glycoproteins found in serum or tissues.
In one embodiment, an acrylate-functionalized poly(ethylene) glycol monomer is used as a hydrogel monomer. For example, the PEG in one embodiment is an acrylate or acrylamide functionalized PEG.
In some embodiments, a hydrogel particle comprises a monofunctional monomer polymerized with at least one bifunctional monomer. One example includes, but is not limited to, the formation of poly-acrylamide polymers using acrylamide and bis-acrylamide (a bifunctional monomer). In another embodiment, a hydrogel particle provided herein comprises a bifunctional monomer polymerized with a second bifunctional monomer. One example include, but is not limited to, the formation of polymers with mixed composition containing compatible chemistries such as acrylamide, bis-acrylamide, and bis-acrylamide structural congeners containing a wide range of additional chemistries. The range of chemically compatible monomers, bifunctional monomers, and mixed compositions is obvious to those skilled in the art and follows chemical reactivity principles know to those skilled in the art. (reference Thermo handbook and acrylamide polymerization handbook). See, for example, the Thermo Scientific Crosslinking Technical Handbook entitled “Easy molecular bonding crosslinking technology,” (available at tools.lifetechnologies.com/content/sfs/brochures/1602163-Crosslinking-Reagents-Handbook.pdf) and the Polyacrylamide Emulsions Handbook (SNF Floerger, available at snf.com.au/downloads/Emulsion_Handbook_E.pdf), the disclosure of each of which is incorporated by reference in its entirety for all purposes.
In one embodiment, a hydrogel particle provided herein comprises a polymerizable monofunctional monomer and is a monofunctional acrylic monomer. Non-limiting examples of monofunctional acrylic monomers for use herein are acrylamide; methacrylamide; N-alkylacrylamides such as N-ethylacrylamide, N-isopropylacrylamide or N-tertbutylacrylamide; N-alkylmethacrylamides such as N-ethylmethacrylamide or Nisopropylmethacrylamide; N,N-dialkylacrylamides such as N,N-dimethylacrylamide and N,N-diethyl-acrylamide; N-[dialkylamino)alkyl]acrylamides such as N-[3dimethylamino) propyl]acrylamide or N-[3-(diethylamino)propyl]acrylamide; N-[(dialkylamino) alkyl]methacrylamides such as N-[3-dimethylamino)propyl]methacrylamide or N-[3-(diethylamino) propyl]methacrylamide; (dialkylamino)alkyl acrylates such as 2-(dimethylamino)ethyl acrylate, 2-(dimethylamino)propyl acrylate, or 2-(diethylamino)ethyl acrylates; and (dialkylamino) alkyl methacrylates such as 2-(dimethylamino) ethyl methacrylate.
A bifunctional monomer is any monomer that can polymerize with a monofunctional monomer of the disclosure to form a hydrogel as described herein that further contains a second functional group that can participate in a second reaction, e.g., conjugation of a fluorophore or cell surface receptor (or domain thereof).
In some embodiments, a bifunctional monomer is selected from the group consisting of: allyl amine, allyl alcohol, allyl isothiocyanate, allyl chloride, and allyl maleimide.
A bifunctional monomer can be a bifunctional acrylic monomer. Non-limiting examples of bifunctional acrylic monomers are N,N′-methylenebisacrylamide, N,N′methylene bismethacrylamide, N,N′-ethylene bisacrylamide, N,N′-ethylene bismethacrylamide, N,N′propylenebisacrylamide and N,N′-(1,2-dihydroxyethylene) bisacrylamide.
Higher-order branched chain and linear co-monomers can be substituted in the polymer mix to adjust the refractive index while maintaining polymer density, as described in U.S. Pat. No. 6,657,030, incorporated herein by reference in its entirety for all purposes.
In some embodiments, a hydrogel comprises a molecule that modulates the optical properties of the hydrogel. Molecules capable of altering optical properties of a hydrogel are discussed further below.
In one embodiment, an individual hydrogel particle or a plurality thereof comprises a biodegradable polymer as a hydrogel monomer. In one embodiment, the biodegradable polymer is a poly(esters) based on polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL), and their copolymers. In one embodiment, the biodegradable polymer is a carbohydrate or a protein, or a combination thereof. For example, in one embodiment, a monosaccharide, disaccharide or polysaccharide, (e.g., glucose, sucrose, or maltodextrin) peptide, protein (or domain thereof) is used as a hydrogel monomer. Other biodegradable polymers include poly(hydroxyalkanoate)s of the PHB-PHV class, additional poly(ester)s, and natural polymers, for example, modified poly(saccharide)s, e.g., starch, cellulose, and chitosan. In another embodiment, the biocompatible polymer is an adhesion protein, cellulose, a carbohydrate, a starch (e.g., maltodextrin, 2-hydroxyethyl starch, alginic acid), a dextran, a lignin, a polyaminoacid, an amino acid, or chitin. Such biodegradable polymers are available commercially, for example, from Sigma Aldrich (St. Louis, Mo.).
The protein in one embodiment comprises only natural amino acids. However, the invention is not limited thereto. For example, self-assembling artificial proteins and proteins with non-natural amino acids (e.g., those incorporated into non-ribosomal peptides or synthetically introduced via synthetic approaches, see for example, Zhang et al. (2013). Current Opinion in Structural Biology 23, pp. 581-587, the disclosure of which is incorporated by reference in its entirety for all purposes), or protein domains thereof, can also be used as hydrogel monomers. The range of non-natural (unnatural) amino acids that can be incorporated into such compositions is well known to those skilled in the art (Zhang et al. (2013). Current Opinion in Structural Biology 23, pp. 581-587; incorporated by reference in its entirety for all purposes). The biodegradable polymer in one embodiment, is used as a co-monomer, i.e., in a mixture of monomers. The biodegradable polymer in one embodiment is a bifunctional monomer.
The biomonomer, in one embodiment, is functionalized with acrylamide or acrylate. For example, in one embodiment, the polymerizable acrylamide functionalized biomolecule is an acrylamide or acrylate functionalized protein (for example, an acrylamide functionalized collagen or functionalized collagen domain), an acrylamide or acrylate functionalized peptide, or an acrylamide or acrylate functionalized monosaccharide, disaccharide or polysaccharide.
Any monosaccharide, disaccharide or polysaccharide (functionalized or otherwise) can be used as a hydrogel monomer. In one embodiment, an acrylamide or acrylate functionalized monosaccharide, disaccharide or polysaccharide is used as a polymerizable hydrogel monomer. In one embodiment, a structural polysaccharide is used as a polymerizable hydrogel monomer. In a further embodiment, the structural polysaccharide is an arabinoxylan, cellulose, chitin or a pectin. In another embodiment, alginic acid (alginate) is used as a polymerizable hydrogel monomer. In yet another embodiment, a glycosaminoglycan (GAG) is used as a polymerizable monomer in the hydrogels provided herein. In a further embodiment, the GAG is chondroitin sulfate, dermatan sulfate, keratin sulfate, heparin, heparin sulfate or hyaluronic acid (also referred to in the art as hyaluron or hyaluronate) is used as a polymerizable hydrogel monomer. The additional range of compatible biomonomers and their reactive chemistries are known be individuals skilled in the art and follow general chemical reactivity principles.
An additional range of biocompatible monomers that can be incorporated are known in the art, see, for example the non-degradable biocompatible monomers disclosed in Shastri (2003). Current Pharmaceutical Biotechnology 4, pp. 331-337, incorporated by reference herein in its entirety for all purposes. Other monomers are provided in de Moraes Porto (2012). Polymer Biocompatibility, Polymerization, Dr. Ailton De Souza Gomes (Ed.), ISBN: 978-953-51-0745-3; InTech, DOI: 10.5772/47786; Heller et al. (2010). Journal of Polymer Science Part A: Polymer Chemistry 49, pp. 650-661; Final Report for Biocompatible Materials (2004), The Board of the Biocompatible Materials and the Molecular Engineering in Polymer Science programmes, ISBN 91-631-4985-0, the disclosure of each of which are hereby incorporated by reference in their entirety.
Biocompatible monomers for use with the hydrogels described herein include in one embodiment, ethyleglycol dimethacrylate (EGDMA), 2-hydroxyethyl methacrylate (HEMA), methylmethacrylte (MMA), methacryloxymethyltrimethylsilane (TMS-MA), N-vinyl-2-pyrrolidon (N-VP), styrene, or a combination thereof.
Naturally occurring hydrogels useful in this invention include various polysaccharides available from natural sources such as plants, algae, fungi, yeasts, marine invertebrates and arthropods. Non-limiting examples include agarose, dextrans, chitin, cellulose-based compounds, starch, derivatized starch, and the like. These generally will have repeating glucose units as a major portion of the polysaccharide backbone. Cross-linking chemistries for such polysaccharides are known in the art, see for example Thermo Scientific Crosslinking Technical Handbook entitled “Easy molecular bonding crosslinking technology,” (available at tools.lifetechnologies.com/content/sfs/brochures/1602163-Crosslinking-Reagents-Handbook.pdf).
Hyaluronan in one embodiment is used as a hydrogel monomer (either as a single monomer or as a co-monomer). Hyaluronan in one embodiment, is functionalized, for example with acrylate or acrylamide. Hyaluronan is a high molecular weight GAG composed of disaccharide repeating units of N-acetylglucosamine and glucuronic acid linked together through alternating β-1,4 and β-1,3 glycosidic bonds. In the human body, hyaluronate is found in several soft connective tissues, including skin, umbilical cord, synovial fluid, and vitreous humor. Accordingly, in one embodiment, where one or more optical properties of a skin cell, umbilical cord cell or vitreous humor cell is desired to be mimicked, in one embodiment, hyaluronan is used as a hydrogel monomer. Methods for fabricating hydrogel particles are described in Xu et al. (2012). Soft Matter. 8, pp. 3280-3294, the disclosure of which is incorporated herein in its entirety for all purposes. As described therein, hyaluronan can be derivatized with various reactive handles depending on the desired cross-linking chemistry and other monomers used to form a hydrogel particle.
In yet other embodiments, chitosan, a linear polysaccharide composed of randomly distributed β-(1-4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit), is used as a hydrogel monomer (either as a single monomer or as a co-monomer).
Other polysaccharides for use as a hydrogel monomer or co-monomer include but are not limited to, agar, agarose, alginic acid, alguronic acid, alpha glucan, amylopectin, amylose, arabinoxylan, beta-glucan, callose, capsullan, carrageenan polysaccharides (e.g., kappa, iota or lambda class), cellodextrin, cellulin, cellulose, chitin, chitosan, chrysolaminarin, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, ficoll, fructan, fucoidan, galactoglucomannan, galactomannan, galactosaminoogalactan, gellan gum, glucan, glucomannan, glucorunoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hypromellose, icodextrin, inulin, kefiran, laminarin, lentinan, levan polysaccharide, lichenin, mannan, mixed-linkage glucan, paramylon, pectic acid, pectin, pentastarch, phytoglycogen, pleuran, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, sinistrin, sizofiran, welan gum, xanthan gum, xylan, xyloglucan, zymosan, or a combination thereof. As described throughout, depending on the desired cross-linking chemistry and/or additional co-monomers employed in the hydrogel, the polysaccharide can be further functionalized. For example, one or more of the polysaccharides described herein in one embodiment is functionalized with acrylate or acrylamide.
In one embodiment, an individual hydrogel particle or a plurality thereof comprises a peptide, protein, a protein domain, or a combination thereof as a hydrogel monomer or plurality thereof. In a further embodiment, the protein is a structural protein, or a domain thereof, for example, such as silk, elastin, titin or collagen, or a domain thereof. In one embodiment, the protein is an extracellular matrix (ECM) component (e.g., collagen, elastin, proteoglycan). In even a further embodiment, the structural protein is collagen. In yet a further embodiment, the collagen is collagen type I, collagen type II or collagen type III or a combination thereof. In another embodiment, the hydrogel monomer comprises a proteoglycan. In a further embodiment, the proteoglycan is decorin, biglycan, testican, bikunin, fibromodulin, lumican, or a domain thereof.
In another embodiment, an acrylate-functionalized structural protein hydrogel monomer is used as a component of the hydrogel provided herein (e.g., an acrylate functionalized protein or protein domain, for example, silk, elastin, titin, collagen, proteoglycan, or a functionalized domain thereof). In a further embodiment, the acrylate functionalized structural protein hydrogel monomer comprises a proteoglycan, e.g., decorin, biglycan, testican, bikunin, fibromodulin, lumican, or a domain thereof.
In one embodiment PEG monomers and oligopeptides can be that mimic extracellular matrix proteins are used in the hydrogels provided herein, for example, with vinyl sulfone-functionalized multiarm PEG, integrin binding peptides and bis-cysteine matrix metalloproteinase peptides as described by Lutolf et al. (2003). Proc. Natl. Acad. Sci. U.S.A. 100, 5413-5418, incorporated by reference in its entirety for all purposes. In this particular embodiment, hydrogels are formed by a Michael-type addition reaction between the di-thiolated oligopeptides and vinyl sulfone groups on the PEG. The range of additional compatible chemistries that can be incorporated here are obvious to those skilled in the art and follow general chemical reactivity principles, see for example Thermo Scientific Crosslinking Technical Handbook entitled “Easy molecular bonding crosslinking technology,” (available at tools.lifetechnologies.com/content/sfs/brochures/1602163-Crosslinking-Reagents-Handbook.pdf).
Other bioactive domains in natural proteins can also be used as a hydrogel monomer or portion thereof. For example, a cell-adhesive integrin binding domain, a controlled release affinity binding domain or a transglutaminase cross-linking domain can be used in the hydrogels provided herein. Details for producing such hydrogels can be found in Martino et al. (2009). Biomaterials 30, 1089; Martino et al. (2011). Sci. Trans. Med. 3, 100ra89; Hu and Messersmith (2003). J. Am. Chem. Soc. 125, 14298, each of which is incorporated by reference in its entirety for all purposes.
In one embodiment, recombinant DNA methods are used to create proteins, designed to gel in response to changes in pH or temperature, for example, by the methods described by Petka et al. (1998). Science 281, pp. 389-392, incorporated by reference in its entirety for all purposes. Briefly, the proteins consist of terminal leucine zipper domains flanking a water-soluble polyelectrolyte segment. In near-neutral aqueous solutions, coiled-coil aggregates of the terminal domains form a three-dimensional hydrogel polymer network.
Common cross linking agents that can be used to crosslink the hydrogels provided herein include but are not limited to ethylene glycol dimethacrylate (EGDMA), tetraethylene glycol dimethacrylate, and N,N′-15 methylenebisacrylamide. The range of additional crosslinking chemistries which can be used are obvious to those skilled in the art and follow general chemical reactivity principles, see for example Thermo Scientific Crosslinking Technical Handbook entitled “Easy molecular bonding crosslinking technology,” (available at tools.lifetechnologies.com/content/sfs/brochures/1602163-Crosslinking-Reagents-Handbook.pdf).
In one embodiment, polymerization of a hydrogel is initiated by a persulfate or an equivalent initiator that catalyzes radical formation. The range of compatible initiators are known to those skilled in the art and follow general chemical reactivity principles, see for example Thermo Scientific Crosslinking Technical Handbook entitled “Easy molecular bonding crosslinking technology,” (available at tools.lifetechnologies.com/content/sfs/brochures/1602163-Crosslinking-Reagents-Handbook.pdf). The persulfate can be any water-soluble persulfate. Non-limiting examples of water soluble persulfates are ammonium persulfate and alkali metal persulfates. Alkali metals include lithium, sodium and potassium. In some embodiments, the persulfate is ammonium persulfate or potassium persulfate. In a further embodiment, polymerization of the hydrogel provided herein is initiated by ammonium persulfate.
Polymerization of a hydrogel can be accelerated by an accelerant which can catalyze the formation of polymerization-labile chemical side groups. The range of possible accelerants is known to those skilled in the art and follow general chemical reactivity principles see for example Thermo Scientific Crosslinking Technical Handbook entitled “Easy molecular bonding crosslinking technology,” (available at tools.lifetechnologies.com/content/sfs/brochures/1602163-Crosslinking-Reagents-Handbook.pdf). The accelerant in one embodiment, is a tertiary amine. The tertiary amine can be any water-soluble tertiary amine. In one embodiment, an accelerant is used in the polymerization reaction and is N,N,N′,N′tetramethylethylenediamine, 3-dimethylamino) propionitrile, or N,N,N′,N′tetramethylethylenediamine (TEMED). In another embodiment, an accelerant is used in the polymerization reaction and isazobis (isobutyronitrile) (AIBN).
As discussed above, the hydrogel for use in the compositions and methods described herein can include any of the monomeric units and crosslinkers as described herein, and in one aspect, are produced as hydrogel particles by polymerizing droplets (see, e.g., FIG. 2). Microfluidic methods of producing a plurality of droplets, including fluidic and rigidified droplets, are known to those of ordinary skill in the art, and described in US Patent Publication No. 2011/0218123 and U.S. Pat. No. 7,294,503, each incorporated herein by reference in their entireties for all purposes. Such methods provide for a plurality of droplets containing a first fluid and being substantially surrounded by a second fluid, where the first fluid and the second fluid are substantially immiscible (e.g., droplets containing an aqueous-based liquid being substantially surrounded by an oil based liquid).
A plurality of fluidic droplets (e.g., prepared using a microfluidic device) may be polydisperse (e.g., having a range of different sizes), or in some cases, the fluidic droplets may be monodisperse or substantially monodisperse, e.g., having a homogenous distribution of diameters, for instance, such that no more than about 10%, about 5%, about 3%, about 1%, about 0.03%, or about 0.01% of the droplets have an average diameter greater than about 10%, about 5%, about 3%, about 1%, about 0.03%, or about 0.01% of the average diameter. The average diameter of a population of droplets, as used herein, refers to the arithmetic average of the diameters of the droplets. Average diameters of the particles can be measured, for example, by light scattering techniques. Average diameters of hydrogel particles in one embodiment, are tailored, for example by varying flow rates of the fluid streams of the first and second fluids within the channel(s) of a microfluidic device, or by varying the volume of the channel(s) of the microfluidic device.
Accordingly, the disclosure provides population of hydrogel particles comprising a plurality of hydrogel particles, wherein the population of hydrogel particles is substantially monodisperse.
The term microfluidic refers to a device, apparatus or system including at least one fluid channel having a cross-sectional dimension of less than 1 mm, and a ratio of length to largest cross-sectional dimension perpendicular to the channel of at least about 3:1. A micro fluidic device comprising a micro fluidic channel is especially well suited to preparing a plurality of mono disperse droplets.
Non-limiting examples of microfluidic systems that may be used with the present invention are disclosed in U.S. Patent Application Publication No. 2006/0163385; U.S. Patent Application Publication No. 2005/0172476; U.S. Patent Application Publication No. 2007/000342; International Patent Application Publication No. WO 2006/096571; U.S. Patent Application Publication No. 2007/0054119; U.S. Pat. No. 7,776,927; and International Patent Application Publication No. WO 2006/078841, each incorporated herein by reference in their entireties for all purposes.
Droplet size is related to microfluidic channel size. The micro fluidic channel may be of any size, for example, having a largest dimension perpendicular to fluid flow of less than about 5 mm or 2 mm, or less than about 1 mm, or less than about 500 μm, less than about 200 μm, less than about 100 μm, less than about 60 μm, less than about 50 μm, less than about 40 μm, less than about 30 μm, less than about 25 μm, less than about 10 μm, less than about 3 μm, less than about 1 μm, less than about 300 nm, less than about 100 nm, less than about 30 nm, or less than about 10 nm.
Droplet size can be tuned by adjusting the relative flow rates. In some embodiments, drop diameters are equivalent to the width of the channel, or within about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% the width of the channel.
The dimensions of a hydrogel particle of the disclosure are substantially similar to the droplet from which it was formed. Therefore, in some embodiments, a hydrogel particle has a diameter of less than about 1 μm, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 600, 800, or less than 1000 μm in diameter. In some embodiments, a hydrogel particle has a diameter of more than about 1 μm, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 600, 800, or greater than 1000 μm in diameter. In one embodiment, a hydrogel particle has a diameter in the range of 5 μm to 100 μm.
In some embodiments, a hydrogel particle of the disclosure is spherical in shape.
In some embodiments, a hydrogel particle of the disclosure does not comprise agarose.
Hydrogel particles in one embodiment, is carried by suspension polymerization, which is also referred to in the art as pearl, bead or granular polymerization (see Elbert (2011). Acta Biomater. 7, pp. 31-56, incorporated by reference herein in its entirety for all purposes). In suspension polymerization, the monomer is insoluble in the continuous phase, for example an aqueous monomer solution in a continuous oil phase. In suspension polymerization, polymerization initiation occurs within the monomer-rich droplets and with greater than one radical per droplet at any time. The monomer phase in one embodiment includes a monomer which can be a bifunctional monomer or a plurality of monomer species (co-monomers, which can be a plurality of bifunctional monomers. The monomer phase in one embodiment, includes an initiator and/or a crosslinking agent.
Emulsion polymerization can also be used to form the hydrogel particles described herein. In emulsion polymerization, the monomer has poor solubility in the continuous phase, similar to suspension polymerization, however, polymerization initiation occurs outside the monomer droplets (see Elbert (2011). Acta Biomater. 7, pp. 31-56, incorporated by reference herein in its entirety for all purposes). In emulsion polymerization embodiments, the initiator causes chain growth of the monomer (or co-monomers) dissolved in the continuous phase or monomer contained in micelles if surfactants are present.
In another embodiment, hydrogel particles are formed by precipitation polymerization, for example as described in Elbert (2011). Acta Biomater. 7, pp. 31-56, incorporated by reference herein in its entirety for all purposes. Precipitation polymerization is a technique that takes advantage of the differences in the solubility of monomer and polymer to produce microparticles. Specifically, it is known that larger polymer chains generally have lower solubility than smaller ones. Accordingly, above a specific molecular weight, phase separation may be favored. Precipitation polymerization initially begins as solution polymerizations in a single phase, homogenous system. Shortly after the start of the polymerization, in one embodiment, a relatively high concentration of polymer chains is present, favoring phase separation by nucleation. As polymerization proceeds, the concentration of polymer chains is low and existing particles capture the chains before nucleation of new particles can occur. Thus, nucleation of particles occurs only for a brief period of time shortly after the start of the reaction, which in one embodiment, results in a narrow size distribution of particles. Additional methods include but are not limited to lithographic particle formation (Helgeson et al. (2011). Curr. Opin. Colloid. Interface Sci. 16, pp. 106-117, incorporated by reference herein in its entirety for all purposes) membrane emulsification (e.g., by the micosieve emulsification technology techniques described by Nanomi B.V. (Netherlands)) and microchannel emulsification (Sugiura et al. (2002). Languimir 18, pp. 5708-5712, incorporated by reference herein in its entirety) and bulk emulsification (SNF Floerger, available at snf.com.au/downloads/Emulsion_Handbook_E.pdf, incorporated by reference herein in its entirety).
In one embodiment, hydrogel particles are formed within a microfluidic device having two oil channels that focus on a central stream of aqueous monomer solution. In this embodiment, droplets form at the interface of the two channels and central stream to break off droplets in water-in-oil emulsion. Once droplets are formed, in one embodiment, they are stabilized prior to polymerization, for example, by adding a surfactant to the oil phase. However, in another embodiment, droplets are not stabilized prior to polymerization. Polymerization of the monomer in one embodiment is triggered by adding an accelerator (e.g., N,N,N′,N′tetramethylethylenediamine) to one or both of the oil channels after initial droplets are formed.
The aqueous monomer solution as provided above can include a single monomer species or a plurality of monomer species. The aqueous monomer solution can include co-monomers, a bifunctional monomer or a combination thereof. In one embodiment, the monomer or plurality of monomers can includes a bifunctional monomer, for example, one of the monomers described above. As described below, co-monomers can be used to modulate forward scatter or side scatter, for example, by adjusting the refractive index of the hydrogel particle.
In one embodiment, the central stream of aqueous monomer solution comprises a cross-linker, for example, N,N′-bisacrylamide. In a further embodiment, the central stream of aqueous monomer solution comprises a cross-linker and an accelerator, in addition to the monomer. In yet a further embodiment, the aqueous monomer solution comprises an initiator, for example an oxidizing agent such as ammonium persulfate.
Forward scatter was modulated by adjusting the refractive index of the gel by adding co-monomers allyl acrylate and allyl methacrylate (see also FIGS. 11 and 12). Forward scatter can also be modulated with side scattering nanoparticles containing sufficient optical resolution/size/density including, but not limited to, higher density colloidal suspensions of silica and/or PMMA particles. Side scattering of the droplets was tuned by adding a colloidal suspension of silica nanoparticles and/or PMMA (poly(methyl methacrylate)) particles ( ̃100 nm) to the central aqueous phase prior to polymerization (FIGS. 11 and 12).
In one embodiment, a bead, plurality of beads, biomolecule, or plurality of biomolecules is embedded (encapsulated) within the hydrogel particle. An encapsulated bead or biomolecule, in one embodiment, is employed to mimic one or more intracellular organelles of a target cell, or a cell after it engulfs a particle. In one embodiment, encapsulating or embedding a bead or biomolecule is accomplished at the time of hydrogel particle formation. For example, beads can be suspended in the appropriate concentration to allow for an average of one bead to be embedded/encapsulated in a single hydrogel particle. The bead suspension can be included, for example, within the aqueous solution of monomer. Similarly, a biomolecule or mixture of biomolecules can be incorporated into the aqueous solution of monomer to encapsulate the biomolecule or biomolecules.
Alternatively, once a hydrogel particle is formed, for example by the methods described above, in one embodiment, it can be further manipulated, for example, by embedding a bead, plurality of beads, biomolecule or plurality of biomolecules within the hydrogel particle.
Accordingly, in one aspect of the invention, a hydrogel comprising an embedded substance is provided.
In one embodiment, the embedded substance is an embedded molecule, for example a biomolecule. The biomolecule can be a single species or a plurality of different species. For example, a protein, peptide, carbohydrate, nucleic acid or combination thereof can be encapsulated within a hydrogel particle of the invention. Moreover, different nucleic acid molecules (e.g., of varying sequences or nucleic acid type such as genomic DNA, messenger RNA or DNA-RNA hybrids) can be encapsulated by the hydrogel particle of the invention. These can be comprised of any protein or nucleic acid as both forms of biological material contain labile chemical side-groups (or can be modified by commercial vendors (e.g., Integrated DNA Technology chemical side group modifications). Such side-groups are compatible with reaction chemistries commonly found in co-monomer compositions (e.g. acrylate chemistry, NETS-ester, primary amines, copper catalyzed click chemistry (Sharpless)). The range of possible embedded molecules which contain compatible chemistries is understood by those skilled in the art.
In one embodiment, different subpopulations of hydrogel particles are fabricated, each with a different concentration of biomolecule. In a further embodiment, the biomolecule is a nucleic acid, a protein, an intracellular ion such as calcium acid (or other biomolecule of the user's choosing, for example, calcium). In another embodiment, different subpopulations of hydrogel particles are fabricated, each with a different concentration of a drug substance. The drug substance in one embodiment is a biomolecule (i.e., a biologic, antibody, antibody drug conjugate, protein/enzyme, peptide, non-ribosomal peptide, or related molecule) or a small molecule synthetic drug (e.g., Type I/II/III polyketide, non-ribosomal peptide with bioactive properties, or other small molecule entity as generally classified by those skilled in the art).
In this regard, the present invention is particularly useful for determining assay resolution where cells are stained for their respective nucleic acid or protein content. In one embodiment, different populations of the hydrogel particles provided herein are encapsulated with known, differing amounts of an intracellular substance, e.g., nucleic acid or protein. Individual hydrogel particles are stained for the intracellular substance and fluorescence is measured via a cytometric device for the individual hydrogels of the various populations. This allows for a generation of a standard curve to establish the sensitivity and dynamic range of the intracellular assay. Once established, a sample can be run through the cytometer to detect target cell(s) if present, and to quantify the amount of intracellular substance in the respective target cell(s). In one embodiment, the embedded substance is an infectious disease biomarker, for example one of the infectious disease biomarkers in the Infectious Disease Biomarker Database (IDBD, see Yang et al. (2008) IDBD: Infectious Disease Biomarker Database. Nucleic Acid Res. 36, pp. D455-D460, incorporated by reference in its entirety for all purposes). In a further embodiment, the infectious disease biomarker is a biomarker of gastrointestinal infection, respiratory infection, neurological infection, urogenital infection, viral infection, hemorrhagic fever, zoonosis, arbovirus, antibiotics resistance or bioterrorism. In a further embodiment, the viral infection is an Ebola infection.
In one embodiment, the methods provided herein are used to determine the sensitivity and/or dynamic range of a cellular nucleic acid quantification assay. In this embodiment, a sample is interrogated for cell types within the sample (if present), and amount of cellular nucleic acid within the cell.
In another embodiment, the present invention provides a means for determining the resolution and/or sensitivity of an intracellular protein quantification assay. Hydrogel particles, in one embodiment, encapsulate known amounts of protein, at various concentrations, and subsequently stained with the appropriate protein antibody. Fluorescence is measured for the various particles to determine the sensitivity and/or dynamic range of the assay. The fluorescence values can then be compared to the values obtained from cells in a sample, to determine whether a target cell is present and whether it contains the intracellular protein, and the amount of the protein.
In one embodiment, individual hydrogel particles are tuned to have at least one optical property substantially similar to a circulating tumor cell or a fetal cell, present in maternal blood. The individual particles are embedded with known quantities of a biomolecule of interest. The particles are used to generate a standard curve for a biomolecule detection assay for the particular cell type.
As provided above, in one aspect of the invention, a hydrogel comprising an embedded substance is provided. In one embodiment, the embedded substance is a bead or plurality of beads. In one embodiment, a hydrogel particle is embedded with a single bead. In another embodiment, individual hydrogels the average number of embedded beads in a plurality of hydrogel particles is one.
In the case where a bead or plurality of beads are embedded into a hydrogel particle, in one embodiment, the optical properties of the bead or plurality of beads are used in combination with the FSC and SSC properties of the hydrogel particle for quality control of a flow cytometry assay. For example, the embedded bead in one embodiment is used as a control to calibrate the flow cytometer system, including the laser source, optics, and stream flow. In another embodiment, the embedded bead is used as a means for quantitating the amount of fluorescence in a sample, e.g., a particular cell. In this regard, embedded beads of various intensities can be used to generate a standard curve of fluorescence to determine whether a cell expresses a certain marker and at what level of expression.
In one embodiment, a bead with the diameter of about 1 μm to about 3 μm, about 2 μm to about 4 μm or about 3 μm to about 7 μm is embedded in a hydrogel provided herein. For example, in one embodiment, the bead has a diameter of about 3 μm to about 3.5 μm. In a further embodiment, the bead is a fluorescent bead. In another embodiment, the bead has a diameter of about 1 μm to about 2.5 μm or about 1.5 μm to about 3 μm. In a further embodiment, the bead is a fluorescent bead and can be stained either internally or at its surface. In even a further embodiment, the fluorescent bead is stained internally. Without wishing to be bound by theory, it is thought that internal staining insulates the fluorophores from environmental interactions that could cause variable fluorescence output.
As provided above, in one embodiment, the embedded bead is a fluorescence bead and in a further embodiment, the fluorescent bead is stained internally. It is within the skill in the art to select the appropriate fluorophore for use in conjunction with an embedded bead. In one embodiment, the bead is derivatized with one or more of the following fluorescent dyes: 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein succinimidylester; 5-(and-6)-carboxyeosin; 5-carboxyfluorescein; 6 carboxyfluorescein; 5-(and-6)-carboxyfluorescein; S-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl)ether,-alanine-carboxamide, or succinimidyl ester; 5-carboxy fluorescein succinimidyl ester; 6-carboxyfluorescein succinimidyl ester; 5-(and-6)-carboxyfluorescein succinimidyl ester; 5-(4,6-dichlorotriazinyl) amino fluorescein; 2 ‘, 7’-difluoro fluorescein; eosin-5-isothiocyanate; erythrosin5-isothiocyanate; 6-(fluorescein-5-carboxamido) hexanoic acid or succinimidyl ester; 6-(fluorescein-5-(and-6)-carboxamido) hexanoic acid or succinimidylester; fluorescein-S-EX succinimidyl ester; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; OregonGreen® 488 carboxylic acid, or succinimidyl ester; Oregon Green® 488 isothiocyanate; Oregon Green® 488-X succinimidyl ester; Oregon Green® 500 carboxylic acid; Oregon Green® 500 carboxylic acid, succinimidylester or triethylammonium salt; Oregon Green® 514 carboxylic acid; Oregon Green® 514 carboxylic acid or succinimidyl ester; RhodamineGreenTM carboxylic acid, succinimidyl ester or hydrochloride; Rhodamine GreenTM carboxylic acid, trifluoroacetamide or succinimidylester; Rhodamine GreenTM-X succinimidyl ester or hydrochloride; RhodolGreenTM carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidylester; bis-(4-carboxypiperidinyl) sulfonerhodamine or di(succinimidylester); 5-(and-6)carboxynaphtho fluorescein, 5-(and-6)carboxynaphthofluorescein succinimidyl ester; 5-carboxyrhodamine 6G hydrochloride; 6-carboxyrhodamine6Ghydrochloride, 5-carboxyrhodamine 6G succinimidyl ester; 6-carboxyrhodamine 6G succinimidyl ester; 5-(and-6)-carboxyrhodamine6G succinimidyl ester; 5-carboxy-2′,4′,5′,7′-tetrabromosulfonefluorescein succinimidyl esteror bis-(diisopropylethylammonium) salt; 5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine; 5-(and-6)-carboxytetramethylrhodamine; 5-carboxytetramethylrhodamine succinimidyl ester; 6-carboxytetramethylrhodaminesuccinimidyl ester; 5-(and -6)-carboxytetramethylrhodamine succinimidyl ester; 6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimidyl ester; 6-carboxy-Xrhodamine succinimidyl ester; 5-(and-6)-carboxy-Xrhodaminesuccinimidyl ester; 5-carboxy-X-rhodamine triethylammonium salt; LissamineTM rhodamine B sulfonyl chloride; malachite green; isothiocyanate; NANOGOLD® mono(sulfosuccinimidyl ester); QSY® 21carboxylic acid or succinimidyl ester; QSY® 7 carboxylic acid or succinimidyl ester; Rhodamine RedTM-X succinimidyl ester; 6-(tetramethylrhodamine-5-(and-6)-carboxamido) hexanoic acid; succinimidyl ester; tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Red® sulfonyl; Texas Red® sulfonyl chloride; Texas Red®-X STP ester or sodium salt; Texas Red®-X succinimidyl ester; Texas Red®-X succinimidyl ester; and X-rhodamine-5-(and-6) isothiocyanate, BODIPY® dyes commercially available from Invitrogen, including, but not limited to BODIPY® FL; BODIPY® TMR STP ester; BODIPY® TR-X STP ester; BODIPY® 630/650-X STPester; BODIPY® 650/665-X STP ester; 6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester; 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-dipropionic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoicacid; 4,4-difluoro-5,7-dimethyl-4-bora3a,4a-diaza-s-indacene-3-pentanoicacid succinimidyl ester; 4,4-difluoro-5,7-dimefhyl-4-bora-3a,4a-diaza-s-indacene-3propionicacid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4adiaza-s-indacene-3-propionicacid succinimidyl ester; 4,4difluoro-5,7-dimefhyl-4-bora-3a,4a-diaza-s-indacene-3propionic acid; sulfosuccinimidyl ester or sodium salt; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3propionyl)amino)hexanoicacid; 6-((4,4-difluoro-5, 7 dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoic acid or succinimidyl ester; N-(4,4-difluoro 5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl) cysteic acid, succinimidyl ester or triethylammonium salt; 6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora3a,4a4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-sindacene-3-propionicacid; 4,4-difluoro-5,7-diphenyl-4-bora3a, 4a-diaza-s-indacene-3-propionicacid succinimidyl ester; 4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid; succinimidyl ester; 6-((4,4-difluoro-5-phenyl-4 bora-3a,4a-diaza-s-indacene-3-propionyl)amino) hexanoicacid or succinimidyl ester; 4,4-difluoro-5-(4-phenyl-1,3butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionicacid succinimidyl ester; 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester; 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl)aminohexanoicacid or succinimidyl ester; 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid; 4, 4-difluoro-5-styryl-4-bora-3a,4a-diaza-sindacene-3-propionic acid; succinimidyl ester; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4adiaza-s-indacene-8-propionicacid; 4,4-difluoro-1,3,5,7-tetramethyl-4bora-3a,4a-diaza-sindacene-8-propionic acid succinimidyl ester; 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-sindacene-3-propionic acid succinimidyl ester; 6-(((4-(4, 4-difluoro-5-(2-thienyl)-4-bora-3a,4adiazas-indacene-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimidyl ester; and 6#(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl) aminohexanoic acid or succinimidyl ester, Alexa fluor dyes commercially available from Invitrogen, including but not limited to Alexa Fluor® 350 carboxylic acid; Alexa Fluor® 430 carboxylic acid; Alexa Fluor® 488 carboxylic acid; Alexa Fluor® 532 carboxylic acid; Alexa Fluor® 546 carboxylic acid; Alexa Fluor® 555 carboxylic acid; Alexa Fluor® 568 carboxylic acid; Alexa Fluor® 594 carboxylic acid; Alexa Fluor® 633 carboxylic acid; Alexa Fluor® 64 7 carboxylic acid; Alexa Fluor® 660 carboxylic acid; and Alexa Fluor® 680 carboxylic acid, cyanine dyes commercially available from Amersham-Pharmacia Biotech, including, but not limited to Cy3 NHS ester; Cy 5 NHS ester; Cy5.5 NHSester; and Cy7 NHS ester.
Other Fluorophores amenable for use with the present invention are provided in Table 2 below.













TABLE 2








Exci-
Emis-




ID
NAME
Alternate Names
tation
sion
Vendor/Source
ACS CAS#





















ISAC148
6-carboxyfluorescein

492
518
PubChem
3301-79-9


ISAC1
6-JOE

520
550
LifeTechnologies
82855-40-1


ISAC2
7-AAD

545
647
LifeTechnologies
7240-37-1


ISAC3
Acridine Orange

503
525
LifeTechnologies
65-61-2


ISAC4
Alexa Fluor 350
AF350; 2H-1-Benzopyran-6-sulfonic acid,
343
442
LifeTechnologies
244636-14-4




7-amino-3-[2-[(2,5-dioxo-1-




pyrrolidinyl)oxy]-2-oxoethyl]-4-methyl-2-




oxo-; 200554-19-4


ISAC6
Alexa Fluor 405
AF405; C46H69N5O15S3
401
425
LifeTechnologies
791637-08-6


ISAC7
Alexa Fluor 430
AF430; C32H42F3N3O9S
433
541
LifeTechnologies
467233-94-9


ISAC8
Alexa Fluor 488
AF488; C25H15Li2N3O13S2
496
519
LifeTechnologies
247144-99-6


ISAC9
Alexa Fluor 500
AF500; CAS#798557-08-1
503
525
LifeTechnologies
798557-08-1


ISAC10
Alexa Fluor 514
AF514; C31H27N3O13S2
517
542
LifeTechnologies
798557-07-0


ISAC11
Alexa Fluor 532
AF532; 1H-Pyrano[3,2-f: 5,6-f′]diindole-
532
553
LifeTechnologies
222159-92-4




10,12-disulfonic acid, 5-[4-[[(2,5-dioxo-1-




pyrrolidinyl)oxy]carbonyl]phenyl]-2,3,7,8-




tetrahydro-2,3,3,7,7,8-hexamethyl-;




271795-14-3


ISAC13
Alexa Fluor 546
AF546; C50H62Cl3N5O14S3
556
573
LifeTechnologies
247145-23-9


ISAC14
Alexa Fluor 555
AF555
555
565
LifeTechnologies
644990-77-2


ISAC15
Alexa Fluor 568
AF568
578
603
LifeTechnologies
247145-38-6


ISAC16
Alexa Fluor 594
AF594
590
617
LifeTechnologies
247145-86-4


ISAC17
Alexa Fluor 610
AF610; C58H77Cl3N6O14S3
612
628
LifeTechnologies
900528-62-3


ISAC18
Alexa Fluor 633
AF633
632
647
LifeTechnologies
477780-06-6


ISAC19
Alexa Fluor 635
AF635
633
647
LifeTechnologies
945850-82-8


ISAC20
Alexa Fluor 647
AF647
650
665
LifeTechnologies
400051-23-2


ISAC21
Alexa Fluor 660
AF660
663
690
LifeTechnologies
422309-89-5


ISAC22
Alexa Fluor 680
AF680
679
702
LifeTechnologies
422309-67-9


ISAC23
Alexa Fluor 700
AF700
702
723
LifeTechnologies
697795-05-4


ISAC24
Alexa Fluor 750
AF750
749
775
LifeTechnologies
697795-06-5


ISAC25
Alexa Fluor 790
AF790
784
814
LifeTechnologies
950891-33-5


ISAC26
AMCA

346
448
SantaCruzBiotech
106562-32-7


ISAC27
AmCyan

457
489
BDBioscences
1216872-44-4


ISAC28
APC
Allophycocyanin
650
660
SigmaAldrich
No names found


ISAC29
APC-Alexa Fluor 680
APC-AF680
655
704
LifeTechnologies
No names found


ISAC30
APC-Alexa Fluor 700
APC-AF700
655
718
LifeTechnologies
No names found


ISAC31
APC-Alexa Fluor 750
APC-AF750
650
775
LifeTechnologies
No names found


ISAC32
APC-Cy5.5
Allophycocyanin-Cy5.5
650
695
LifeTechnologies
No names found


ISAC33
APC-Cy7
Allophycocyanin-Cy7
650
767
LifeTechnologies
No names found


ISAC34
APC-eFluor 750
eFluor750APC
650
750
eBioscience
No names found


ISAC35
APC-eFluor 780
eFluor780APC
650
780
eBioscience
1472056-77-1


ISAC36
APC-H7
H7APC
650
765
BDBioscences
1366000-62-5


ISAC37
APC-Vio770
Vio770APC
652
775
Miltenyl Biotech
No names found


ISAC38
Atto488

501
523
ATTO-TEC
923585-42-6


ISAC39
BIOTIN

0
0
PubChem
58-85-5


ISAC40
BODIPY FL

502
511
SantaCruzBiotech
165599-63-3


ISAC41
BODIPY R6G
4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-
527
547
LifeTechnologies
335193-70-9




s-indacene-3-propionic acid, succinimidyl




ester; C22H18BF2N3O4


ISAC43
Brilliant Violet 421
BV421
406
423
Biolegend
1428441-68-2


ISAC44
Brilliant Violet 510
BV510
405
510
Biolegend
No names found


ISAC45
Brilliant Violet 570
BV570
407
571
Biolegend
1428441-76-2


ISAC46
Brilliant Violet 605
BV605
407
603
Biolegend
1632128-60-9


ISAC47
Brilliant Violet 612
BV612
0
0
Biolegend
1428441-91-1


ISAC48
Brilliant Violet 650
BV650
407
647
Biolegend
No names found


ISAC49
Brilliant Violet 711
BV711
405
711
Biolegend
No names found


ISAC50
Brilliant Violet 785
BV785
405
786
Biolegend
1613592-44-1


ISAC53
Calcein
CAS#: 1461-15-0
493
514
LifeTechnologies
1461-15-0


ISAC51
Calcein AM

496
517
PubChem
148504-34-1


ISAC52
Calcein Blue AM

360
445
PubChem
168482-84-6


ISAC54
Calcein Violet AM

400
452
LifeTechnologies
No names found


ISAC55
Calcium Sensor Dye eFluor

490
514
eBioscience
No names found



514


ISAC56
Cascade Blue

401
420
PubChem
1325-87-7


ISAC57
Cascade Yellow

400
550
Synchem UG & Co.
220930-95-0







KG


ISAC58
Cell Proliferation Dye

405
445
eBioscience
No names found



eFluor 450


ISAC59
Cell Proliferation Dye

652
672
eBioscience
No names found



eFluor 670


ISAC60
CellTrace Violet Cell

392
455
LifeTechnologies
No names found



Proliferation


ISAC61
CellVue Claret

655
657
SigmaAldrich
1042142-46-0


ISAC62
CFSE

492
525
SantaCruzBiotech
150347-59-4


ISAC63
CPC
◯□cresolphthalein complexone
488
660
Chemical Book
2411-89-4


ISAC65
Cy2

492
507
GElifesciences
102185-03-5


ISAC66
Cy3

552
566
GElifesciences
146368-16-3


ISAC67
Cy3.5

581
598
GElifesciences
189767-45-1


ISAC68
Cy5

633
670
GElifesciences
144377-05-9


ISAC69
Cy5.5

677
695
GElifesciences
210892-23-2


ISAC70
Cy7

743
767
GElifesciences
169799-14-8


ISAC71
Cychrome

565
667
BDBioscences
245670-67-1


ISAC73
CyQUANT DNA

502
522
LifeTechnologies
No names found


ISAC74
CyTRAK Orange
1,5-bis{[2-(di-methylamino)
514
609
Abcam
1195771-25-5




ethyl]amino}-4,8-dihydroxyanthracene-


(eBioscience)




9,10-dione


ISAC76
DAPI

358
462
PubChem
47165-04-8


ISAC77
DCFH

505
525
SigmaAldrich
106070-31-9


ISAC79
DiA
DiA; 4-Di-16-ASP (4-(4-
455
586
LifeTechnologies
371114-38-4




(Dihexadecylamino)styryl)-N-




Methylpyridinium Iodide); C46H79IN2


ISAC81
DiD
DiD′ solid; DiIC18(5) solid (1,1′-
647
669
LifeTechnologies
127274-91-3




Dioctadecyl-3,3,3′,3′-




Tetramethylindodicarbocyanine, 4-




Chlorobenzenesulfonate Salt);




C67H103ClN2O3S


ISAC84
DiI
DiI Stain (1,1′-Dioctadecyl-3,3,3′,3′-
550
568
LifeTechnologies
41085-99-8




Tetramethylindocarbocyanine Perchlorate




(‘DiI’; DiIC18(3))); C59H97ClN2O4; 3H-




Indolium, 2-(3-(1,3-dihydro-3,3-dimethyl-




1-octadecyl-2H-indol-2-ylidene)-1 -




propenyl)-3,3-dimethyl-1-octadecyl-,




perchlorate/


ISAC88
DiO
DiO′; DiOC18(3) (3,3′-
489
506
LifeTechnologies
34215-57-1




Dioctadecyloxacarbocyanine




Perchlorate); C53H85ClN2O6;




Benzoxazolium, 3-octadecyl-2-[3-(3-




octadecyl-2(3H)-benzoxazolylidene)-1-




propenyl]-, perchlorate/


ISAC92
DiR
DiR′; DiIC18(7) (1,1′-Dioctadecyl-3,3,3′,3′-
750
781
LifeTechnologies
100068-60-8




Tetramethylindotricarbocyanine Iodide);




C63H101IN2


ISAC95
DRAQ5

645
683
CellSignallingTech
254098-36-7


ISAC96
DRAQ7

599
694
CellSignallingTech
1533453-55-2


ISAC97
DsRED

532
595
Clontech
469863-23-8


ISAC98
dsRed2-RFP

555
582
Clontech
No names found


ISAC99
DY547
547 Dyomics
557
574
Dynomics
947138-67-2


ISAC100
DY634
634 Dyomics
635
658
Dynomics
1189010-49-8


ISAC101
DY647
647 Dyomics
650
665
Dynomics
890317-39-2


ISAC102
DyLight 350
DL350
353
432
PierceNet
1436849-83-0


ISAC103
DyLight 405
DL405
400
420
PierceNet
1051927-09-3


ISAC104
DyLight 488
DL488
493
518
PierceNet
1051927-12-8


ISAC105
DyLight 549
DL549
562
576
JacksonImmunoRes
1051927-13-9


ISAC106
DyLight 550
DL550
562
576
PierceNet
1340586-78-8


ISAC107
DyLight 594
DL594
593
618
PierceNet
1268612-00-5


ISAC108
DyLight 633
DL633
638
658
PierceNet
1051927-14-0


ISAC109
DyLight 649
DL649
654
670
JacksonImmunoRes
1051927-15-1


ISAC110
DyLight 650
DL650
652
672
PierceNet
1364214-13-0


ISAC111
DyLight 680
DL680
682
712
PierceNet
1051927-24-2


ISAC112
DyLight 800
DL800
777
794
PierceNet
1051927-23-1


ISAC113
EB
Ethidium Bromide
523
604
SigmaAldrich
1239-45-8


ISAC114
ECD

563
613
LifeTechnologies
88475-75-6


ISAC116
ECFP
enhanced cyan fluorescent protein
435
477
MyBiosource
No names found


ISAC118
EdU
EdU(5-ethynyl-2\u2032-deoxyuridine);
0
0
LifeTechnologies
61135-33-9




C11H12N2O5


ISAC120
EdU Alexa Fluor 488

496
516
LifeTechnologies
No names found


ISAC121
EdU Alexa Fluor 647

650
665
LifeTechnologies
No names found


ISAC122
EdU Pacific Blue

405
455
LifeTechnologies
No names found


ISAC123
eFluor 450

400
450
eBioscience
1592653-87-6


ISAC124
eFluor 450 Fixable Viability

400
450
eBioscience
No names found



Dye


ISAC125
eFluor 490

350
490
eBioscience
No names found


ISAC126
eFluor 506 Fixable Viability

420
506
eBioscience
No names found



Dye


ISAC127
eFluor 525

350
525
eBioscience
No names found


ISAC128
eFluor 565

350
565
eBioscience
No names found


ISAC129
eFluor 585

350
604
eBioscience
No names found


ISAC130
eFluor 605

350
605
eBioscience
1248429-27-7


ISAC131
eFluor 615

590
622
eBioscience
No names found


ISAC132
eFluor 625

350
625
eBioscience
No names found


ISAC133
eFluor 650

350
650
eBioscience
No names found


ISAC134
eFluor 660

633
658
eBioscience
1634649-16-3


ISAC135
eFluor 670

0
0
eBioscience
1437243-07-6


ISAC136
eFluor 700

350
700
eBioscience
No names found


ISAC137
eFluor 710

350
710
eBioscience
No names found


ISAC138
eFluor 780 Fixable Viability

755
780
eBioscience
No names found



Dye


ISAC139
EGFP
enhanced green fluorescent protein
480
510
MyBiosource
No names found


ISAC141
Emerald 300

289
530
LifeTechnologies
No names found


ISAC142
Eosin

525
546
SigmaAldrich
17372-87-1


ISAC143
Ethidium Homodimer-1

528
617
SigmaAldrich
61926-22-5


ISAC144
Ethidium Monoazide EMA

510
590
SigmaAldrich
58880-05-0


ISAC145
EYFP
enhanced yellow fluorescent protein
515
528
MyBiosource
No names found


ISAC147
FAM

492
518
PubChem
76823-03-5


ISAC149
FITC
Fluorescein
500
520
PubChem
27072-45-3


ISAC153
Fluo-3
C51H50Cl2N2O23; Glycine, N-[4-[6-
506
526
LifeTechnologies
123632-39-3




[(acetyloxy)methoxy]-2,7-dichloro-3-oxo-




3H-xanthen-9-yl]-2-[2-[2-[bis[2-




[(acetyloxy)methoxy]-2-oxyethyl]amino]-5-




methylphenoxy]ethoxy]phenyl]-N-[2-




[(acetyloxy)methoxy]-2-oxyethyl]-,




(acetyloxy)methyl ester/


ISAC155
Fluo-4
C51H50F2N2O23; Glycine, N-[4-[6-
494
516
LifeTechnologies
273221-59-3




[(acetyloxy)methoxy]-2,7-difluoro-3-oxo-




3H-xanthen-9-yl]-2-[2-[2-[bis[2-




[(acetyloxy)methoxy]-2-oxoethyl]amino]-




5-methylphenoxy]ethoxy]phenyl]-N-[2-




[(acetyloxy)methoxy]-2-oxoethyl]-,




(acetyloxy)methyl ester/


ISAC152
FLMA
Fluorescein-5-maleimide
495
520
PierceNet
75350-46-8


ISAC157
Fluoro-Emerald
Dextran, Fluorescein, 10,000 MW,
495
523
LifeTechnologies
194369-11-4




Anionic, Lysine Fixable


ISAC159
Fura Red



LifeTechnologies
149732-62-7


ISAC162
Fura3
Fura-2 LeakRes (AM)
325
510
SigmaAldrich
172890-84-5


ISAC164
FxCycle Far Red

640
658
LifeTechnologies
No names found


ISAC165
FxCycle Violet
C16H17Cl2N5; 1H-Indole-6-
358
462
LifeTechnologies
28718-90-3




carboximidamide, 2-[4-




(aminoiminomethyl)phenyl]-,




dihydrochloride/


ISAC167
GFP
green fluorescent protein
488
515
MyBiosource
No names found


ISAC169
GFP Violet Excited

398
515
MyBiosource
No names found


ISAC170
GFP-Vex1

398
515
MyBiosource
No names found


ISAC171
HiLyte Fluor 488

501
527
Anaspec
1051927-29-7


ISAC172
HiLyte Fluor 555

550
566
Anaspec
1051927-30-0


ISAC173
HiLyte Fluor 647

649
674
Anaspec
925693-87-4


ISAC174
HiLyte Fluor 680

0
0
Anaspec
1051927-34-4


ISAC175
HiLyte Fluor 750

754
778
Anaspec
1051927-32-2


ISAC176
Hoechst 33258

345
455
SigmaAldrich
23491-45-4


ISAC177
Hoechst 33342
bisBenzimide H 33342 trihydrochloride
343
455
SigmaAldrich
23491-52-3


ISAC179
Hydroxycoumarin
C10H6O5; 7-hydroxycoumarin-3-
360
450
LifeTechnologies
43070-85-5




carboxylic acid; 2H-1-Benzopyran-3-




carboxylic acid, 7-hydroxy-2-oxo-/; 4-




chloromethyl-7-hydroxycoumarin


ISAC183
Indo-1
Indo-1 AM Calcium Sensor Dye;
347
480
LifeTechnologies
96314-96-4




C47H51N3O22; 1H-Indole-6-carboxylic




acid, 2-[4-[bis[2-[(acetyloxy)methoxy]-2-




oxoethyl]amino]-3-[2-[2-[bis[2-




[(acetyloxy)methoxy]-2-oxoetyl]amino]-5-




methylphenoxy]ethoxy]phenyl]-,




(acetyloxy)methyl ester/


ISAC187
JC-1
5,5′,6,6′-tetrachloro-1,1′,3,3′-
593
595
LifeTechnologies
3520-43-2




tetraethylbenzimidazolylcarbocyanine




iodide; C25H27Cl4IN4


ISAC189
Krome Orange

398
530
Beckman Coulter
1558035-65-6


ISAC190
Leadmium

490
520
LifeTechnologies
No names found


ISAC191
LIVE/DEAD Fixable Aqua
Aqua LIVE/DEAD
367
526
LifeTechnologies
No names found



Dead Cell Stain


ISAC193
LIVE/DEAD Fixable Blue
Blue LIVE/DEAD
343
442
LifeTechnologies
No names found



Dead Cell Stain


ISAC195
LIVE/DEAD Fixable Far Red

650
670
LifeTechnologies
No names found



Dead Cell Stain


ISAC196
LIVE/DEAD Fixable Green
Green LIVE/DEAD
498
525
LifeTechnologies
No names found



Dead Cell Stain


ISAC198
LIVE/DEAD Fixable Near-IR

752
776
LifeTechnologies
No names found



Dead Cell Stain


ISAC199
LIVE/DEAD Fixable Red

594
612
LifeTechnologies
No names found



Dead Cell Stain


ISAC200
LIVE/DEAD Fixable Violet
Violet LIVE/DEAD
403
455
LifeTechnologies
No names found



Dead Cell Stain


ISAC202
LIVE/DEAD Fixable Yellow
Yellow LIVE/DEAD
401
551
LifeTechnologies
No names found



Dead Cell Stain


ISAC204
Lucifer Yellow
C13H9Li2N5O9S2; 1H-
428
544
LifeTechnologies
82446-52-4




Benz[de]isoquinoline-5,8-disulfonic acid,




6-amino-2-[(hydrazinocarbonyl)amino]-




2,3-dihydro-1,3-dioxo-, dilithium salt/


ISAC206
Magnesium Green
C33H17Cl2K5N2O13; Glycine, N-[2-
507
531
LifeTechnologies
170516-41-3




(carboxymethoxy)-4-[[(2′,7′-dichloro-3′,6′-




dihydroxy-3-oxospiro[isobenzofuran-




1(3H),9′-[9H]xanthen]-5-




yl)carbonyl]amino]phenyl]-N-




(carboxymethyl)-, pentapotassium salt/


ISAC208
Marina Blue
C16H11F2NO7; 2,5-Pyrrolidinedione, 1-
364
461
LifeTechnologies
215868-23-8




[[(6,8-difluoro-7-hydroxy-4-methyl-2-oxo-




2H-1-benzopyran-3-yl)acetyl]oxy]-/;


ISAC210
mBanana

540
553
Clontech
1114839-40-5


ISAC211
mCherry

587
610
Clontech
1628764-31-7


ISAC212
mCitrine

516
529
Not Commercialized
1357606-54-2


ISAC213
MethylCoumarin
AMCA-X, SE (6-((7-Amino-4-
360
448
LifeTechnologies
1333-47-7




Methylcoumarin-3-Acetyl)amino)Hexanoic




Acid, Succinimidyl Ester); C22H25N3O7


ISAC216
MitoTracker Green
C34H28Cl5N3O; Benzoxazolium, 2-[3-
490
512
LifeTechnologies
1304563-13-0




[5,6-dichloro-1,3-bis[[4-




(chloromethyl)phenyl]methyl]-1,3-dihydro-




2H-benzimidazol-2-ylidene]-1-propenyl]-




3-methyl-, chloride/


ISAC218
MitoTracker Orange
C24H24Cl2N2O
550
575
LifeTechnologies
No names found


ISAC219
MitoTracker Red
C39H36Cl5N3
578
598
LifeTechnologies
No names found


ISAC220
mOrange

548
562
Clontech
1114839-60-9


ISAC221
mPlum

590
649
Clontech
1399820-93-9


ISAC222
mRaspberry

597
624
Clontech
1452799-41-5


ISAC223
mRFP1

584
607
Not Commercialized
1452799-30-2


ISAC224
mStrawberry

574
596
Clontech
1114834-99-9


ISAC225
Na-Green
Sodium Green TM,
506
532
LifeTechnologies
195244-55-4




tetra(tetramethylammonium) salt;




C84H100Cl4N8O19


ISAC228
Nile Red
C20H18N2O2; 5H-
559
637
LifeTechnologies
7385-67-3




Benzo[\u03B1]phenoxazin-5-one, 9-




(diethylamino)-/


ISAC230
Oregon Green

491
519
LifeTechnologies
195136-58-4


ISAC232
Oregon Green 488-X,

500
525
LifeTechnologies
890416-18-9



succinimidyl ester


ISAC233
Oregon Green 514
Oregon Green ® 514 carboxylic acid,
510
532
LifeTechnologies
198139-53-6




succinimidyl ester; C26H12F5NO9S


ISAC235
Pacific Blue
PacBlue; Pacific Blue TMsuccinimidyl
405
455
LifeTechnologies
215868-31-8




ester; C14H7F2NO7


ISAC236
Pacific Blue succinimidyl

405
455
LifeTechnologies
215868-33-0



ester


ISAC237
Pacific Orange
PacOrange
403
551
LifeTechnologies
1122414-42-9


ISAC240
PE-Alexa Fluor 610
RPE-AF610
563
628
LifeTechnologies
No names found


ISAC241
PE-Alexa Fluor 647
RPE-AF647
567
669
LifeTechnologies
No names found


ISAC242
PE-Alexa Fluor 680
RPE-AF680
570
702
LifeTechnologies
No names found


ISAC243
PE-Alexa Fluor 700
RPE-AF700
563
720
LifeTechnologies
No names found


ISAC244
PE-Alexa Fluor 750
RPE-AF750
570
776
AbD Serotec
No names found


ISAC245
PE-CF594
PE-Dazzle 594
564
612
BDBioscences
1613592-67-8


ISAC72
PE-Cy5

565
667
BDBioscences
1448849-77-1


ISAC248
PE-Cy5.5

563
695
AbD Serotec
No names found


ISAC249
PE-Cy7

563
760
AbD Serotec
1429496-42-3


ISAC250
PE-DY590

563
599
LSBio
No names found


ISAC251
PE-DY647

563
672
LSBio
No names found


ISAC252
PerCP

490
675
AbD Serotec
422551-33-5


ISAC253
PerCP-Cy5.5

488
695
AbD Serotec
1474026-81-7


ISAC254
PerCP-eFluor 710

488
710
eBioscience
1353683-31-4


ISAC115
PE-Texas Red

563
613
LifeTechnologies
No names found


ISAC256
PE-Vio770

565
775
Miltenyl Biotech
No names found


ISAC257
pHrodo
pHrodo TM Red, succinimidyl ester
560
586
LifeTechnologies
No names found




(pHrodo TM Red, SE); pHrodo TM Green




STP Ester


ISAC260
pHrodo Green STP Ester

560
586
LifeTechnologies
No names found


ISAC258
pHrodo Red, succinimidyl

560
586
LifeTechnologies
No names found



ester


ISAC261
Phycocyanin

617
646
SigmaAldrich
11016-15-2


ISAC262
PicoGreen
Quant-iT TM PicoGreen ® dsDNA Reagent
502
522
LifeTechnologies
177571-06-1


ISAC264
PKH2
PKH2 Green Fluorescent Cell Linker
490
504
SigmaAldrich
145687-07-6


ISAC266
PKH26
PKH26 Red Fluorescent Cell Linker
551
567
SigmaAldrich
154214-55-8


ISAC268
PKH67
PKH67 Green Fluorescent Cell Linker
490
504
SigmaAldrich
257277-27-3


ISAC270
POPO-1
C41H54I4N6O2; Benzoxazolium, 2,2′-
433
457
LifeTechnologies
169454-15-3




[1,3-propanediylbis[(dimethyliminio)-3,1-




propanediyl-1(4H)-pyridinyl-4-




ylidenemethylidyne]]bis[3-methyl]-,




tetraiodide/


ISAC272
PO-PRO-1
C20H27I2N3O; Benzoxazolium, 3-
435
457
LifeTechnologies
157199-56-9




methyl-2-[[1-[3-




(trimethylammonio)propyl]-4(1H)-




pyridinylidene]methyl]-, diiodide/;


ISAC274
Propidium Iodide
C27H34I2N4; Phenanthridinium, 3,8-
350
617
LifeTechnologies
25535-16-4




diamino-5-[3-




(diethylmethylammonio)propyl]-6-phenyl-,




diiodide


ISAC276
PURE

0
0
Not Commercialized
No names found


ISAC277
Pyronin Y

547
560
SigmaAldrich
92-32-0


ISAC278
Qdot 525

350
525
LifeTechnologies
885332-45-6


ISAC279
Qdot 545

350
545
LifeTechnologies
948906-89-6


ISAC280
Qdot 565

350
565
LifeTechnologies
859509-02-7


ISAC281
Qdot 585

350
585
LifeTechnologies
885332-46-7


ISAC282
Qdot 605

350
605
LifeTechnologies
849813-89-4


ISAC283
Qdot 625

350
625
LifeTechnologies
1144512-19-5


ISAC284
Qdot 655

350
655
LifeTechnologies
674287-64-0


ISAC285
Qdot 705

350
705
LifeTechnologies
885332-47-8


ISAC286
Qdot 800

350
800
LifeTechnologies
885332-50-3


ISAC287
RD1
R-Phycoerythrin
563
578
LifeTechnologies
1376573-14-6


ISAC295
Rhodamine

550
570
LifeTechnologies
No names found


ISAC290
Rho 110
Rhodamine 110
497
520
LifeTechnologies
13558-31-1


ISAC293
Rho 123
Rhodamine 123
507
529
LifeTechnologies
62669-70-9


ISAC296
Rhodamine Green
Rhodamine Green TMcarboxylic acid,
505
527
LifeTechnologies
189200-71-3




succinimidyl ester, hydrochloride;




C25H18ClN3O7












ISAC297
Rhodamine Green carboxylic acid, succinimidyl ester, hydrochloride
505
527
LifeTechnologies
254732-34-8













ISAC298
Rhodamine Red

573
591
LifeTechnologies
99752-92-8


ISAC299
Rhodamine Red-X
Rhodamine Red TM-X, succinimidyl ester;
570
576
LifeTechnologies
178623-12-6




C37H44N4O10S2


ISAC300
Rhodamine Red-X,

570
576
LifeTechnologies
178623-13-7



succinimidyl ester


ISAC301
RiboFlavin

266
531
SigmaAldrich
83-88-5


ISAC239
R-Phycoerythrin
PE
563
578
LifeTechnologies
11016-17-4












ISAC303
SNARF-1 carboxylic acid, acetate, succinimidyl ester
549
586
LifeTechnologies
No names found













ISAC302
SNARF-1 pH 6
SNARF ®-1 carboxylic acid, acetate,
549
586
LifeTechnologies
No names found




succinimidyl ester; C33H24N2O9


ISAC304
SNARF-1 pH 9

576
640
LifeTechnologies
No names found


ISAC305
Spectral Red

506
665
MyBiosource
No names found


ISAC306
SureLight P1

545
667
Abcam (Columbia
No names found







Biosciences)


ISAC307
SureLight P3

614
662
Abcam
1365659-06-8


ISAC308
SureLight PBXL-3

614
662
Abcam
No names found


ISAC309
SYBR Green

498
522
SigmaAldrich
217087-73-5


ISAC310
SYTO 11

506
526
LifeTechnologies
173080-67-6


ISAC311
SYTO 13

488
506
LifeTechnologies
173080-69-8


ISAC312
SYTO 16

488
520
LifeTechnologies
173080-72-3


ISAC313
SYTO 17

618
637
LifeTechnologies
189233-66-7


ISAC314
SYTO 45

450
486
LifeTechnologies
335078-86-9


ISAC315
SYTO 59

622
643
LifeTechnologies
235422-34-1


ISAC316
SYTO 60

650
681
LifeTechnologies
335079-14-6


ISAC317
SYTO 61

618
651
LifeTechnologies
335079-15-7


ISAC318
SYTO 62

650
681
LifeTechnologies
286951-08-4


ISAC319
SYTO 82

540
560
LifeTechnologies
335079-10-2


ISAC320
SYTO 9

482
500
LifeTechnologies
208540-89-0


ISAC321
SYTOX AADvanced

546
646
LifeTechnologies
No names found


ISAC322
SYTOX Blue

431
480
LifeTechnologies
396077-00-2


ISAC323
SYTOX Green

504
523
LifeTechnologies
194100-76-0


ISAC324
SYTOX Orange

547
570
LifeTechnologies
324767-53-5


ISAC325
SYTOX Red

640
658
LifeTechnologies
915152-67-9


ISAC326
tdTomato

554
581
Clontech
1114838-94-6


ISAC334
Tetramethylrhodamine
TMRho
553
581
LifeTechnologies
70281-37-7


ISAC329
Texas Red
Texas Red ®-X, succinimidyl ester;
589
615
LifeTechnologies
82354-19-6




C41H44N4O10S2


ISAC330
Texas Red-X, succinimidyl

589
615
LifeTechnologies
216972-99-5



ester


ISAC331
Thiazole Orange

500
530
SigmaAldrich
107091-89-4


ISAC332
ThiolTracker Violet

406
526
LifeTechnologies
No names found


ISAC335
TO-PRO-1
TO-PRO ®-1 iodide (515/531);
509
533
LifeTechnologies
157199-59-2




C24H29I2N3S; Quinolinium, 4-[(3-




methyl-2(3H)-




benzothiazolylidene)methyl]-1-[3-




(trimethylammonio)propyl]-, diiodide/;


ISAC338
TO-PRO-3
TO-PRO ®-3 iodide (642/661);
642
661
LifeTechnologies
157199-63-8




C26H31I2N3S; Quinolinium, 4-[3-(3-




methyl-2(3H)-benzothiazolylidene)-1-




propenyl]-1-[3-




(trimethylammonio)propyl]-, diiodide/


ISAC341
TOTO-1
TOTO ®-1 iodide (514/533);
509
533
LifeTechnologies
143413-84-7




C49H58I4N6S2; Quinolinium, 1-1′-[1,3-




propanediylbis[(dimethyliminio)-3,1-




propanediyl]]bis[4-[(3-methyl-2(3H)-




benzothiazolylidene)methyl]]-,




tetraiodide/


ISAC344
TOTO-3
TOTO ®-3 iodide (642/660);
642
661
LifeTechnologies
166196-17-4




C53H62I4N6S2


ISAC346
Tricolor

563
670
LifeTechnologies
478184-50-8


ISAC347
TRITC
Tetramethylrhodamine;
547
572
LifeTechnologies
745735-42-6




tetramethylrhodamine-5-(and-6)-




isothiocyanate; C25H21N3O3S;




Xanthylium, 9-(2-




carboxyisothiocyanatophenyl)-3,6-




bis(dimethylamino)-, inner salt/


ISAC351
TruRed

490
695
Not Commercialized
396076-95-2


ISAC352
V19

397
572
Not Commercialized
No names found


ISAC353
V450

405
448
BDBioscences
1257844-82-8


ISAC354
V500

415
500
BDBioscences
1333160-12-5


ISAC355
VioBlue

400
452
Miltenyl Biotech
1431147-59-9


ISAC356
VioGreen

388
520
Miltenyl Biotech
No names found


ISAC357
Vybrant DyeCycle Green

505
535
LifeTechnologies
1431152-50-9


ISAC358
Vybrant DyeCycle Orange

518
563
LifeTechnologies
1055990-89-0


ISAC359
Vybrant DyeCycle Ruby

637
686
LifeTechnologies
1345202-72-3


ISAC360
Vybrant DyeCycle Violet

370
436
LifeTechnologies
1015439-88-9


ISAC361
YFP
Yellow Fluorescent Protein
505
530
Clontech
No names found


ISAC363
YO-PRO-1
YO-PRO ®-1 iodide (491/509);
491
506
LifeTechnologies
152068-09-2




C24H29I2N3O


ISAC365
YO-PRO-3
YO-PRO ®-3 iodide (612/631);
613
629
LifeTechnologies
157199-62-7




C26H31I2N3O; Quinolinium, 4-[3-(3-




methyl-2(3H)-benzoxazolylidene)-1-




propenyl]-1-[3-




(trimethylammonio)propyl]-, diiodide/


ISAC368
YOYO-1
YOYO ®-1 iodide (491/509);
491
509
LifeTechnologies
143413-85-8




C49H58I4N6O2;


ISAC370
YOYO-3
YOYO ®-3 iodide (612/631);
613
629
LifeTechnologies
156312-20-8




C53H62I4N6O2; Quinolinium, 1,1′-[1,3-




propanediylbis[(dimethyliminio)-3,1-




propanediyl]]bis[4-[3-(3-methyl-2(3H)-




benzoxazolylidene)-1-propenyl]]-,




tetraiodide/;


ISAC373
ZsGreen

494
517
Clontech
1216871-88-3









Commercially available beads including, but not limited to, those sold by Bangs Laboratories, Inc, Sperhotech Inc., Thermo Scientific, Inc. and equivalent suppliers) can be used in combination with the hydrogel particles described herein. Depending on the assay, it is within the ordinary skill in the art to select a bead with the proper bead diameter, fluorescent emission and/or excitation spectrum and/or fluorescent intensity. For example, a quality control bead used in conjunction with a blue, red or UV laser can be embedded into one or more hydrogel particles provided herein. For example, an AlignflowTM flow cytometry alignment bead for blue lasers (catalog no. A-16500 (2.5 μm), A-16503 (6.0 μm)), red lasers (catalog no. A-16501 (2.5 μm), A-16504 (6.0 μm)) or UV lasers (catalog no. A-16502 (2.5 μm), A-16505 (6.0 μm)) can be embedded in on or more of the hydrogel particles provided herein.
In one embodiment, a fluorescent bead that can be excited at any wavelength from 365 nm-650 nm is embedded in a hydrogel particle. In one embodiment, the bead is a “rainbow particle” that contains a mixture of fluorophores, for example 4 fluorophores, 5 fluorophores, 6 fluorophores, seven fluorophores or eight fluorophores. In this regard, the user selects which wavelength to excite the particle, depending on the fluorophore being interrogated. Rainbow particles are commercially available, for example, from BD Biosciences (catalog nos. 556298 (mid range FL1 fluorescence), 556286 (6 color, 3.0-3.4 μm), 556288 (6 color, 6.0-6.4 μm), 559123 (8 color)) and Spherotech in various diameters (e.g., catalog nos. RCP20-5 (4 color), RCP-30-5 (6 peaks), RCP-30-5A (8 peaks)
A cell sorting set-up bead can be embedded in one or more of the hydrogel particles provided herein. In one embodiment, a cell sorting set-up beads approximates the size, emission wavelength, and intensity of a biological sample, and can be used to calibrate a flow cytometer's cell sorting system, including laser source, optics, and stream flow. In one embodiment, a cell sorting set-up beads is embedded in one or more hydrogel particles and is amenable for use with a UV, blue, green/yellow or red laser. Where a green laser is used, in one embodiment, the embedded bead is excited at 570 nm with emission of 575 nm, but may also be exited at 488 nm. Commercially available cell sorting set-up beads are available, for example, from Life Technologies (catalog nos. C-16506 (UV laser), C-16508 (blue laser), C-16509 (green-yellow laser), C-16507 (red laser)).
A compensation control bead can also be embedded in one or more of the hydrogel particles provided herein. Accurate compensation is an important parameter for effective multicolor analysis in flow cytometry. However, cellular-based compensation controls are not completely effective as many antigens are not highly expressed, and dimly stained cells can lead to inaccurate compensation settings.
A compensation control bead, in one embodiment, includes a fluorescent antibody conjugate capture capacity (positive compensation bead) or is inert (negative compensation bead). The compensation bead is mixed with a fluorophore-conjugated human, mouse, rat, hamster, or rabbit antibody; the two components provide a distinct high-signal positive control with an appropriate negative population that can then be used to set compensation properly regardless of the intensity of the cells in the actual experiment. Once the antibody is mixed with the bead, it is embedded in one or more of the hydrogel particles provided herein. Commercially available compensation beads are available, for example, from Life Technologies (catalog nos. A-10344, A-10389, A10497, A10513) and Spherotech (catalog nos. CMIg-P-08-2K, CMIg-P-30-2K, CMIg-P-50-3K, CMIg-P-70-3K).
In one embodiment, a hydrogel particle with an embedded/encapsulated bead is used as a reference for a cellular assay, for example, a phagocytosis assay cytoxicity assay, motility assay, viability assay, etc. Phagocytosis is the process by which a cell engulfs a solid particle to form an internal vesicle known as a phagosome. In this regard, a hydrogel particle can be tuned to have one or more optical properties substantially similar to a phagocyte, before and after the phagocyte engulfs a particle. Accordingly, in one embodiment, the hydrogel particles provided herein are used as control particles for a phagocytosis assay. In a further embodiment, (i) one or more of the optical properties of a hydrogel particle is substantially similar to a phagocyte prior to particle uptake and (ii) one or more of the optical properties of a second hydrogel particle is substantially similar to a phagocyte after to particle uptake. In this regard, a control is generated for measuring particle uptake by a phagocyte.
In one embodiment, the phagocyte is a professional phagocyte. In another embodiment, the phagocyte is a non-professional phagocyte (i.e., a cell that consumes dying cells and foreign organisms). In a further embodiment, the non-professional phagocyte is an epithelial cell, endothelial cell, fibroblast or mesenchymal cell. Hydrogel particles in one embodiment, are tuned to have one or more optical properties substantially similar to a professional phagocyte set forth in Table 3 below (prior to and/or after particle uptake).








TABLE 3





Location
Phagocyte type







Blood
Neutrophil, monocyte


Bone marrow
Macrophage, monocyte, sinusoidal cell, lining cell


Bone tissue
Osteoclast


Gut and intestinal
Macrophage


Peyer's patches


Connective tissue
Histiocyte, macrophage, monocyte, dendritic cell


Liver
Kupffer cell, monocyte


Lung
Self-replicating macrophage, monocyte, mast cell,



dendritic cell


Lymphoid tissue
Free and fixed macrophages and monocytes, dendritic



cell


Nervous tissue
Microglial cell (CD4+)


Spleen
Free and fixed macrophages, monocytes, sinusoidal



cell


Thymus
Free and fixed macrophages, monocytes


Skin
Resident Langerhans cells, dendritic cells,



conventional macrophage, mast cell









In one embodiment, a plurality of hydrogel particles of the invention, embedded with a substance such as nucleic acid or a bead is used as control reagents for a genomic cytometry assay. In this regard, a specific number of copies of a particular chromosome, RNA sequence and/or DNA sequence can be mimicked by the embedded substance. The hydrogel particle can then be used as a control for a sample being probed for genetic information, such as the number of copies of a chromosome, the number of copies of an RNA sequence and/or the number of copies of an RNA sequence.
The three primary modes of deconvolution for flow cytometry are the two passive optical properties of a particle (forward scattering, FSC, corresponding to the refractive index, or RI; and side scattering, SSC) and biomarkers present on the surface of a given cell type. Therefore, compositions that allow hydrogel particles of the disclosure to mimic specific cell types with respect to these three modes are useful for providing synthetic, robust calibrants for flow cytometry.
In one embodiment, the refractive index (RI) of a disclosed hydrogel particle is greater than about 1.10, greater than about 1.15, greater than about 1.20, greater than about 1.25, greater than about 1.30, greater than about 1.35, greater than about 1.40, greater than about 1.45, greater than about 1.50, greater than about 1.55, greater than about 1.60, greater than about 1.65, greater than about 1.70, greater than about 1.75, greater than about 1.80, greater than about 1.85, greater than about 1.90, greater than about 1.95, greater than about 2.00, greater than about 2.10, greater than about 2.20, greater than about 2.30, greater than about 2.40, greater than about 2.50, greater than about 2.60, greater than about 2.70, greater than about 2.80, or greater than about 2.90.
In another embodiment, the refractive index (RI) of a disclosed hydrogel particle is about 1.10 to about 3.0, or about 1.15 to about 3.0, or about 1.20 to about 3.0, or about 1.25 to about 3.0, or about 1.30 to about 3.0, or about 1.35 to about 3.0, or about 1.4 to about 3.0, or about 1.45 to about 3.0, or about 1.50 to about 3.0, or about 1.6 to about 3.0, or about 1.7 to about 3.0, or about 1.8 to about 3.0, or about 1.9 to about 3.0, or about 2.0 to about 3.0.
In some embodiments, the refractive index (RI) of a disclosed hydrogel particle is less than about 1.10, less than about 1.15, less than about 1.20, less than about 1.25, less than about 1.30, less than about 1.35, less than about 1.40, less than about 1.45, less than about 1.50, less than about 1.55, less than about 1.60, less than about 1.65, less than about 1.70, less than about 1.75, less than about 1.80, less than about 1.85, less than about 1.90, less than about 1.95, less than about 2.00, less than about 2.10, less than about 2.20, less than about 2.30, less than about 2.40, less than about 2.50, less than about 2.60, less than about 2.70, less than about 2.80, or less than about 2.90.
The SSC of a disclosed hydrogel particle is most meaningfully measured in comparison to that of target cell. In some embodiments, a disclosed hydrogel particle has an SSC within 30%, within 25%, within 20%, within 15%, within 10%, within 5%, or within 1% that of a target cell, as measured by a cytometric device.
The SSC of a hydrogel particle in one embodiment, is modulated by incorporating a high-refractive index molecule (or plurality thereof) in the hydrogel. In one embodiment, a high-refractive index molecule is provided in a hydrogel particle, and in a further embodiment, the high-refractive index molecule is colloidal silica, alkyl acrylate, alkyl methacrylate or a combination thereof. Thus in some embodiments, a hydrogel particle of the disclosure comprises alkyl acrylate and/or alkyl methacrylate. Concentration of monomer in one embodiment is adjusted to further adjust the refractive index of the hydrogel particle.
Alkyl acrylates or Alkyl methacrylates can contain 1 to 18, 1 to 8, or 2 to 8, carbon atoms in the alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tertbutyl, 2-ethylhexyl, heptyl or octyl groups. The alkyl group may be branched or linear.
High-refractive index molecules can also include vinylarenes such as styrene and methylstyrene, optionally substituted on the aromatic ring with an alkyl group, such as methyl, ethyl or tert-butyl, or with a halogen, such as chlorostyrene.
In some embodiments, FSC is modulated by adjusting the percentage of monomer present in the composition thereby altering the water content present during hydrogel formation. In one embodiment, where a monomer and co-monomer are employed, the ratio of monomer and co-monomer is adjusted to change the hydrogel particle's forward scatter properties. This is shown in both FIG. 11 and FIG. 12.
The FSC of a disclosed hydrogel particle is most meaningfully measured in comparison to that of target cell. In some embodiments, a disclosed hydrogel particle has an FSC within 30%, within 25%, within 20%, within 15%, within 10%, within 5%, or within 1% that of a target cell, as measured by a cytometric device.
FSC is related to particle volume, and thus can be modulated by altering particle diameter, as described herein. Generally, it has been observed that large objects refract more light than smaller objects leading to high forward scatter signals (and vice versa). Accordingly, particle diameter in one embodiment is altered to modulate FSC properties of a hydrogel particle. For example, hydrogel particle diameter is increased in one embodiment is altered by harnessing larger microfluidic channels during particle formation.
SSC can be engineered by encapsulating nanoparticles within hydrogels to mimic organelles in a target cell. In some embodiments, a hydrogel particle of the disclosure comprises one or more types of nanoparticles selected from the group consisting of: polymethyl methacrylate (PMMA) nanoparticles, polystyrene (PS) nanoparticles, and silica nanoparticles. See also FIGS. 11 and 12 which show that addition of various concentrations of nanoparticles allow for the adjustment of side scatter of a particle. Without wishing to be bound by theory, the ability to selectively tune both forward and side scatter of a hydrogel, as described herein, allows for a robust platform to mimic a vast array of cell types.
Although the invention is mainly described with respect to the modification of optical properties, the invention is not limited thereto. For example, hydrogel particles can be fabricated and adjusted to tune the capacitance of the particles, e.g., to calibrate coulter counters. In one embodiment, a hydrogel particle's capacitance is adjusted by altering the amount of hydrogel monomer in the composition. For example, polyanaline, polyacetylene; polyphenylene vinylene; polypyrrole (X═NH) and polythiophene (X═S) co-monomers; and polyaniline (X═NH/N) and polyphenylene sulfide (X═S) co-monomer concentrations can all be adjusted to alter capacitance. In one embodiment, the concentration of one or more of these monomers is increased to increase the capacitance of the hydrogel particle.
In some embodiments, a hydrogel particle of the disclosure has material modulus properties (e.g., elasticity) more closely resembling that of a target cell as compared to a polystyrene bead of the same diameter.
After the hydrogel particle is formed, one or more of the particle's surfaces can be functionalized, for example, to mimic one or more optical properties of a target cell or a labeled target cell. The functionalized hydrogel particle can also include an embedded bead or substance such as a biomolecule, as described above. In one embodiment, one or more hydrogel particles are functionalized with one or more fluorescent dyes, one or more cell surface markers (or epitope binding regions thereof), or a combination thereof. In one embodiment, the hydrogel particle is formed by polymerizing at least one bifunctional monomer and after formation, the hydrogel particle includes one or more functional groups that can be used for further attachment of a cell surface marker, an epitope binding region of a cell surface marker, a fluorescent dye, or combination thereof. The free functional group, in one embodiment, is an amine group, a carboxyl group, a hydroxyl group or a combination thereof. Depending on the functionalization desired, it is to be understood that multiple bifunctional monomers can be used, for example, to functionalize the particle using different chemistries and with different molecules.
A hydrogel particle can be functionalized with any fluorescent dye known in the art, including fluorescent dyes listed in The MolecularProbes® Handbook-A Guide to Fluorescent Probes and Labeling Technologies, incorporated herein by reference in its entirety for all purposes. Functionalization can be mediated by a compound comprising a free amine group, e.g. allylamine, which can be incorporated into a bifunctional monomer used to form the hydrogel, as discussed above.
Non-limiting examples of known fluorescent dyes that can be used to functionalize the surface of a hydrogel particle described herein include: 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein succinimidylester; 5-(and-6)-carboxyeosin; 5-carboxyfluorescein; 6 carboxyfluorescein; 5-(and-6)-carboxyfluorescein; S-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl)ether,-alanine-carboxamide, or succinimidyl ester; 5-carboxyfluoresceinsuccinimidyl ester; 6-carboxyfluorescein succinimidyl ester; 5-(and-6)-carboxyfluorescein succinimidyl ester; 5-(4,6-dichlorotriazinyl) amino fluorescein; 2 ‘, 7’-difluoro fluorescein; eosin-5-isothiocyanate; erythrosin5-isothiocyanate; 6-(fluorescein-5-carboxamido) hexanoic acid or succinimidyl ester; 6-(fluorescein-5-(and-6)-carboxamido) hexanoic acid or succinimidylester; fluorescein-S-EX succinimidyl ester; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; OregonGreen® 488 carboxylic acid, or succinimidyl ester; Oregon Green® 488 isothiocyanate; Oregon Green® 488-X succinimidyl ester; Oregon Green® 500 carboxylic acid; Oregon Green® 500 carboxylic acid, succinimidylester or triethylammonium salt; Oregon Green® 514 carboxylic acid; Oregon Green® 514 carboxylic acid or succinimidyl ester; RhodamineGreenTM carboxylic acid, succinimidyl ester or hydrochloride; Rhodamine GreenTM carboxylic acid, trifluoroacetamide or succinimidylester; Rhodamine GreenTM-X succinimidyl ester or hydrochloride; RhodolGreenTM carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidylester; bis-(4-carboxypiperidinyl) sulfonerhodamine or di(succinimidylester); 5-(and-6)carboxynaphtho fluorescein, 5-(and-6)carboxynaphthofluorescein succinimidyl ester; 5-carboxyrhodamine 6G hydrochloride; 6-carboxyrhodamine6Ghydrochloride, 5-carboxyrhodamine 6G succinimidyl ester; 6-carboxyrhodamine 6G succinimidyl ester; 5-(and-6)-carboxyrhodamine6G succinimidyl ester; 5-carboxy-2′,4′,5′,7′-tetrabromosulfonefluorescein succinimidyl esteror bis-(diisopropylethylammonium) salt; 5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine; 5-(and-6)-carboxytetramethylrhodamine; 5-carboxytetramethylrhodamine succinimidyl ester; 6-carboxytetramethylrhodaminesuccinimidyl ester; 5-(and -6)-carboxytetramethylrhodamine succinimidyl ester; 6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimidyl ester; 6-carboxy-Xrhodamine succinimidyl ester; 5-(and-6)-carboxy-Xrhodaminesuccinimidyl ester; 5-carboxy-X-rhodamine triethylammonium salt; LissamineTM rhodamine B sulfonyl chloride; malachite green; isothiocyanate; NANOGOLD® mono(sulfosuccinimidyl ester); QSY® 21carboxylic acid or succinimidyl ester; QSY® 7 carboxylic acid or succinimidyl ester; Rhodamine RedTM-X succinimidyl ester; 6-(tetramethylrhodamine-5-(and-6)-carboxamido) hexanoic acid; succinimidyl ester; tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Red® sulfonyl; Texas Red® sulfonyl chloride; Texas Red®-X STP ester or sodium salt; Texas Red®-X succinimidyl ester; Texas Red®-X succinimidyl ester; and X-rhodamine-5-(and-6) isothiocyanate.
Other examples of fluorescent dyes for use with the hydrogel particles described herein include, but are not limited to, BODIPY® dyes commercially available from Invitrogen, including, but not limited to BODIPY® FL; BODIPY® TMR STP ester; BODIPY® TR-X STP ester; BODIPY® 630/650-X STPester; BODIPY® 650/665-X STP ester; 6-dibromo-4, 4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester; 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-dipropionic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoicacid; 4,4-difluoro-5,7-dimethyl-4-bora3a,4a-diaza-s-indacene-3-pentanoicacid succinimidyl ester; 4,4-difluoro-5,7-dimefhyl-4-bora-3a,4a-diaza-s-indacene-3propionicacid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4adiaza-s-indacene-3-propionicacid succinimidyl ester; 4,4difluoro-5,7-dimefhyl-4-bora-3a,4a-diaza-s-indacene-3propionic acid; sulfosuccinimidyl ester or sodium salt; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3propionyl)amino) hexanoic acid; 6-((4,4-difluoro-5, 7 dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino) hexanoic acid or succinimidyl ester; N-(4, 4-difluoro 5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl) cysteic acid, succinimidyl ester or triethylammonium salt; 6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora3a, 4a4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-sindacene-3-propionicacid; 4,4-difluoro-5, 7-diphenyl-4-bora3a, 4a-diaza-s-indacene-3-propionic acid succinimidyl ester; 4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid; succinimidyl ester; 6-((4,4-difluoro-5-phenyl-4 bora-3a,4a-diaza-s-indacene-3-propionyl)amino) hexanoicacid or succinimidyl ester; 4,4-difluoro-5-(4-phenyl-1,3butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionicacid succinimidyl ester; 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester; 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl)aminohexanoicacid or succinimidyl ester; 4,4-difluoro-5-styryl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid; 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-sindacene-3-propionic acid; succinimidyl ester; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4adiaza-s-indacene-8-propionicacid; 4,4-difluoro-1,3,5,7-tetramethyl-4bora-3a,4a-diaza-sindacene-8-propionicacid succinimidyl ester; 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-sindacene-3-propionicacid succinimidyl ester; 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4adiazas-indacene-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimidyl ester; and 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl) styryloxy)acetyl) aminohexanoic acid or succinimidyl ester.
Fluorescent dyes for derivatization of the surface of one or more hydrogel particles in one embodiment, include, but are not limited to, Alexa fluor dyes commercially available from Invitrogen, including but not limited to Alexa Fluor® 350 carboxylic acid; Alexa Fluor® 430 carboxylic acid; Alexa Fluor® 488 carboxylic acid; Alexa Fluor® 532 carboxylic acid; Alexa Fluor® 546 carboxylic acid; Alexa Fluor® 555 carboxylic acid; Alexa Fluor® 568 carboxylic acid; Alexa Fluor® 594 carboxylic acid; Alexa Fluor® 633 carboxylic acid; Alexa Fluor® 64 7 carboxylic acid; Alexa Fluor® 660 carboxylic acid; and Alexa Fluor® 680 carboxylic acid. In another embodiment, fluorescent dyes for use with the hydrogel particles and methods described herein include cyanine dyes commercially available from Amersham-Pharmacia Biotech, including, but not limited to Cy3 NHS ester; Cy 5 NHS ester; Cy5.5 NHSester; and Cy7 NHS ester.
It is within the ordinary skill in the art to select a suitable dye or dyes based on the desired spectral excitation and emission properties of the hydrogel particle.
Hydrogel particles, in one embodiment, are functionalized with one or more cell surface markers (see, e.g., Tables 4 and 7-8), or fragments thereof, for example, extracellular portions thereof in the case of transmembrane proteins, for example, by attaching the one or more cell surface markers, extracellular portions or ligand binding regions thereof to the particle via a free amine, free carboxyl and/or free hydroxyl group present on the surface of the hydrogel particle. Functionalization of a hydrogel particle with a dye or cell surface molecule can also occur through a linker, for example a streptavidin/biotin conjugate.
Depending on the target cell, individual hydrogel particles can be derivatized with one or more cell surface markers, or fragments thereof, for example, extracellular portions thereof in the case of transmembrane proteins to further mimic the structural properties of the target cell. Tables 4 and 7-8, provided below, sets forth a non-limiting list of cell surface markers that can be used to derivative hydrogel particles, depending on the target cell. Although the cell surface marker is provided, it is understood that a portion of the cell surface marker, for example, a receptor binding portion, a ligand binding portion, or an extracellular portion of the marker can be used to derivative the hydrogel particle (at the free functional group, as described above). See also FIGS. 11 and 12 which show that hydrogel surface modification with for example, a cell surface receptor, together with the selective tuning of FSC and/or SSC, allows for the fabrication of a hydrogel particle with the desired feature(s).









TABLE 4





Target Cell
Cell Surface Marker(s) (human)
Cell Surface Marker(s) (mouse)







B Cell
CD19, CD20
CD19, CD22 (B cell activation




marker), CD45R/B220


T Cell
CD3, CD4, CD8
CD3, CD4, CD8


Activated T Cells
CD25, CD69
CD25, CD69


Dendritic Cell
CD1c, CD83, CD123, CD141,
CD11c, CD123, MHC II



CD209, MHC II


Plasmacytoid
CD123, CD303, CD304
CD11cint, CD317


Dendritic Cells*


Platelet (resting)
CD42b
CD41


Platelet (activated)
CD62P
CD62P


Natural Killer Cells
CD16, CD56
CD49b (clone DX5)


Hematopoietic Stem
CD34, CD90
CD48, CD117, CD150, Sca-1


Cell


Macrophage
CD11b, CD68, CD163
F4/80, CD68


Monocyte
CD14, CD16, CD64
CD11b, CD115, Ly-6C


Plasma Cell
CD138
CD138


Red Blood Cell
CD235a
TER-119


Neutrophil
CD15, CD16
CD11b, Ly-6B.2, Ly6G, Gr-1


Basophil
2D7 antigen, CD123, CD203c,
CD200R3, FcεRIα



FcεRIα


Eosinophil
CD11b, CD193, EMR1, Siglec-8
CD11b, CD193, F4/80, Siglec-F


Granulocyte
CD66b
CD66b, Gr-1/Ly6G, Ly6C


Endothelial cell
CD146
CD146 MECA-32, CD106, CD31,




CD62E (activated endothelial cell)


Epithelial cell
CD326
CD326 (EPCAM1)


Natural Killer (NK)
CD56
CD335 (NKp46)


cell


Myeloid derived
CD11b, CD14, CD33 (Siglec-3)
CD11b, GR1


suppressor cell


(MDSC)









Cell types including but not limited to various cell lines such as CHO, HEK-293, BHK-21, NS0, MDCK, VERO, MRC-S, W1-38 and Sp2/0 Mouse Myeloma (hybridomas). Table 5 and Table 6 each provides other cell types for use with the hydrogel particles described herein.







TABLE 5







keratinocyte of epidermis


basal cell of epidermis


keratinocyte of fingernails and toenails


basal cell of nail bed


hair shaft cells


medullary hair shaft cells


cortical hair shaft cells


cuticular hair shaft cells


hair-root sheath cells


cuticular hair-root sheath cells


hair-root sheath cells of Huxley's layer


hair-root sheath cells of Henle's layer


external hair-root sheath cells


hair matrix cell (stem cell)


surface epithelial cell of stratified squamous epithelium of tongue


surface epithelial cell of stratified squamous epithelium of oral cavity


surface epithelial cell of stratified squamous epithelium of esophagus


surface epithelial cell of stratified squamous epithelium of anal canal


surface epithelial cell of stratified squamous epithelium of distal urethra


surface epithelial cell of stratified squamous epithelium of vagina


basal cell of these epithelia


cell of urinary epithelium


cells of salivary gland


Mucous cells of salivary gland


Serous cell of salivary gland


cell of von Ebner's gland in tongue


cell of mammary gland


cell of lacrimal gland


cell of ceruminous gland of ear


cell of eccrine sweat gland


cell of eccrine sweat gland


cell of apocrine sweat gland


cell of gland of Moll in eyelid


cell of sebaceous gland


cell of Bowman's gland in nose


cell of Brunner's gland in duodenum


cell of seminal vesicle


cell of prostate gland


cell of bulbourethral gland


cell of Bartholin's gland


cell of gland of Littre


cell of endometrium of uterus


isolated goblet cell of respiratory and digestive tracts


mucous cell of lining of stomach


zymogenic cell of gastric gland


oxyntic cell of gastric gland


acinar cell of pancreas


Paneth cell of small intestine


type II pneumocyte of lung


Clara cell of lung


cells of anterior pituitary


cell of intermediate pituitary


cells of posterior pitutiary


cells of gut and respiratory tract


cells of thyroid gland


cells of parathyroid gland


cells of adrenal gland


steroid hormones


cells of gonads


cells of juxtaglomerular apparatus of kidney


juxtaglomerular cell


macula


densa cell


peripolar cell


mesangial cell


brush border cell of intestine


striated duct cell of exocrine glands


gall bladder epithelial cell


brush border cell of proximal tubule of kidney


distal tubule cell of kidney


nonciliated cell of ductulus efferens


epididymal principal cell


epididymal basal cell


hepatocyte


white fat cell


brown fat cell


lipocyte of liver


type I pneumocyte


pancreatic duct cell


parietal cell of kidney glomerulus


podocyte of kidney glomerulus


cell of thin segment of loop of Henle


collecting duct cell (in kidney)


duct cell of seminal vesicle


duct cell of prostate gland


vascular endothelial cells of blood vessels and lymphatics


fenestrated vascular endothelial cells


continuous vascular endothelial cells


splenic vascular endothelial cells


synovial cell


serosal cell


squamous cell lining perilymphatic space of ear


cells lining endolymphatic space of ear


squamous cell


columnar cells of endolymphatic sac


“dark” cell


vestibular membrane cell


stria vascularis basal cell


stria vascularis marginal cell


cell of Claudius


cell of Boettcher


choroid plexus cell


squamous cell of pia-arachnoid


cells of ciliary epithelium of eye


corneal “endothelial” cell


Ciliated Cells of respiratory tract


Ciliated Cells of oviduct and of endometrium of uterus


Ciliated Cells of rete testis and ductulus efferens


Ciliated Cells of central nervous system


epithelial


ameloblast


nonepithelial


chondrocytes


osteoblast/osteocyte


osteoprogenitor cell


hyalocyte of vitreous body of eye


stellate cell of perilymphatic space of ear


skeletal muscle cells


heart muscle cells


smooth muscle cells (various)


myoepithelial cells


red blood cell


megakaryocyte


macrophages and related cells


neutrophil


eosinophil


basophil


mast cell


T lymphocyte


B lymphocyte


photoreceptors (rods, cones, and can be blue sensitive, green sensitive,


red sensitive)


inner hair cell of organ of Corti


outer hair cell of organ of Corti


type I hair cell of vestibular apparatus of ear


type II hair cell of vestibular apparatus of ear


type II taste bud cell


olfactory neuron


basal cell of olfactory epithelium


carotid body cell type I


carotid body cell type II


Merkel cell of epidermis


primary sensory neurons specialized for touch (various)


primary sensory neurons specialized for temperature - cold sensitive


primary sensory neurons specialized for temperature - heat sensitive


primary sensory neurons specialized for pain (various)


proprioceptive primary sensory neurons (various)


Autonomic Neurons


inner pillar cell


outer pillar cell


inner phalangeal cell


outer phalangeal cell


border cell


Hensen cell


supporting cell of vestibular apparatus


supporting cell of taste bud (type I taste bud cell)


supporting cell of olfactory epithelium


Schwann cell


satellite cell (encapsulating peripheral nerve cell bodies)


enteric glial cell


neurons


glial cells


anterior lens epithelial cell


lens fiber (crystallin-containing cell)


melanocyte


retinal pigmented epithelial cell


oogonium/oocyte


spermatocyte


spermatogonium (stem cell for spermatocyte)


ovarian follicle cell


Sertoli cell (in testis)


thymus epithelial cell


Salivary gland mucous cell


Salivary gland number 1


Von Ebner's gland cell in tongue


Mammary gland cell


Lacrimal gland cell


Ceruminous gland cell in ear


Eccrine sweat gland dark cell


Eccrine sweat gland clear cell


Apocrine sweat gland cell


Gland of Moll cell in eyelid


Sebaceous gland cell


Bowman's gland cell in nose


Brunner's gland cell in duodenum


Seminal vesicle cell


Prostate gland cell


Bulbourethral gland cell


Bartholin's gland cell


Gland of Littre cell


Uterus endometrium cell


goblet cell of respiratory and digestive tracts


Stomach lining mucous cell


Gastric gland zymogenic cell


Gastric gland oxyntic cell


Pancreatic acinar cell


Paneth cell of small intestine


pneumocyte of lung


Clara cell of lung


anterior pituitary cells


Somatotropes


Lactotropes


Thyrotropes


Gonadotropes


Corticotropes


melanocyte-stimulating hormone


Magnocellular neurosecretory cells secreting:


Gut and respiratory tract cells secreteing:


Thyroid gland cells


thyroid epithelial cell


parafollicular cell


Parathyroid gland cells


Parathyroid chief cell


Oxyphil cell


Adrenal gland cells


chromaffin cells


secreting steroid hormones (mineralcorticoids and gluco corticoids)


Leydig cell of testes secreting testosterone


Theca interna cell of ovarian follicle secreting estrogen


Corpus luteum cell of ruptured ovarian follicle secreting progesterone


Granulosa lutein cells


Theca lutein cells


Juxtaglomerular cell (renin secretion)


Macula densa cell of kidney


Peripolar cell of kidney


Mesangial cell of kidney


epidermal keratinocyte


Epidermal basal cell


Keratinocyte of fingernails and toenails


Nail bed basal cell (stem cell)


Medullary hair shaft cell


Cortical hair shaft cell


Cuticular hair shaft cell


Cuticular hair root sheath cell


Hair root sheath cell of Huxley's layer


Hair root sheath cell of Henle's layer


External hair root sheath cell


Hair matrix cell (stem cell)


epithelial cell of stratified squamous epithelium of cornea,


epithelial cell of stratified squamous epithelium of tongue


epithelial cell of stratified squamous epithelium of oral cavity


epithelial cell of stratified squamous epithelium of esophagus


epithelial cell of stratified squamous epithelium of anal canal


epithelial cell of stratified squamous epithelium of distalurethra


epithelial cell of stratified squamous epithelium of vagina


basal cell (stem cell) of epithelia of cornea


basal cell (stem cell) of epithelia of tongue


basal cell (stem cell) of epithelia of oral cavity


basal cell (stem cell) of epithelia of esophagus


basal cell (stem cell) of epithelia of anal canal


basal cell (stem cell) of epithelia of distal urethra


basal cell (stem cell) of epithelia of vagina


Urinary epithelium cell


Auditory inner hair cell of organ of Corti


Auditory outer hair cell of organ of Corti


basal cell of olfactory epithelium


Cold-sensitive primary sensory neurons


Heat-sensitive primary sensory neurons


Merkel cell of epidermis (touch sensor)


Olfactory receptor neuron


Pain-sensitive primary sensory neurons (various types)


Photoreceptor cells of retina in eye:


Photoreceptor rod cells


Photoreceptor blue-sensitive cone cell of eye


Photoreceptor green-sensitive cone cell of eye


Photoreceptor red-sensitive cone cell of eye


Proprioceptive primary sensory neurons


Touch-sensitive primary sensory neurons


Type I carotid body cell


Type II carotid body cell


Type I hair cell of vestibular system of ear


Type II hair cell of vestibular system of ear


Type I taste bud cell


Cholinergic neural cell


Adrenergic neural cell


Peptidergic neural cell


Inner pillar cell of organ of Corti


Outer pillar cell of organ of Corti


Inner phalangeal cell of organ of Corti


Outer phalangeal cell of organ of Corti


Border cell of organ of Corti


Hensen cell of organ of Corti


Vestibular apparatus supporting cell


Taste bud supporting cell


Olfactory epithelium supporting cell


Schwann cell


Satellite glial cell


Enteric glial cell


Astrocyte


Neuron cells


Oligodendrocyte


Spindle neuron


Anterior lens epithelial cell


Crystallin-containing lens fiber cell


Hepatocyte


Adipocytes (white fat cell, brown fat cell, liver lipocyte)


Kidney parietal cell


Kidney glomerulus podocyte


Kidney proximal tubule brush border cell


Loop of Henle thin segment cell


Kidney distal tubule cell


Kidney collecting duct cell


Type I pneumocyte


Pancreatic duct cell


Nonstriated duct cell


principal cell


Intercalated cell


Duct cell


Intestinal brush border cell


Exocrine gland striated duct cell


Gall bladder epithelial cell


Ductulus efferens nonciliated cell


Epididymal principal cell


Epididymal basal cell


Ameloblast epithelial cell


Planum semilunatum epithelial cell of vestibular system of ear


Organ of Corti interdental epithelial cell


Loose connective tissue fibroblasts


Corneal fibroblasts (corneal keratocytes)


Tendon fibroblasts


Bone marrow reticular tissue fibroblasts


nonepithelial fibroblasts


Pericyte


Nucleus pulposus cell of intervertebral disc


Cementoblast/cementocyte


Odontoblast/odontocyte


Hyaline cartilage chondrocyte


Fibrocartilage chondrocyte


Elastic cartilage chondrocyte


Osteoblast/osteocyte


Osteoprogenitor cell


Hyalocyte of vitreous body of eye


Stellate cell of perilymphatic space of ear


Hepatic stellate cell (Ito cell)


Pancreatic stelle cell


skeletal muscle Cell


Red skeletal muscle cell (slow)


White skeletal muscle cell (fast)


Intermediate skeletal muscle cell


nuclear bag cell of muscle spindle


nuclear chain cell of muscle spindle


Satellite cell (stem cell)


Heart muscle cells


Ordinary heart muscle cell


Nodal heart muscle cell


Purkinje fiber cell


Smooth muscle cell


Myoepithelial cell of iris


Myoepithelial cell of exocrine glands


Erythrocyte


Megakaryocyte


Monocyte


Connective tissue macrophage


Epidermal Langerhans cell


Osteoclast (in bone)


Dendritic cell (in lymphoid tissues)


Microglial cell (in central nervous system)


Neutrophil granulocyte


Eosinophil granulocyte


Basophil granulocyte


Hybridoma cell


Mast cell


Helper T cell


Suppressor T cell


Cytotoxic T cell


Natural Killer T cell


B cell


Natural killer cell


Reticulocyte


Stem cells and committed progenitors for the blood and immune


system (various types)


Oogonium/Oocyte


Spermatid


Spermatocyte


Spermatogonium cell


Spermatozoon


Ovarian follicle cell


Sertoli cell (in testis)


Thymus epithelial cell


Interstitial kidney cells
















TABLE 6







Keratinizing Epithelial Cells


keratinocyte of epidermis (=differentiating epidermal cell)


basal cell of epidermis (stem cell)


keratinocyte of fingernails and toenails


basal cell of nail bed (stem cell)


hair shaft cells


medullary


cortical


cuticular


hair-root sheath cells


Cuticular root sheath cells


root sheath cells of Huxley's layer


root sheath cells of Henle's layer


external root sheath cells


hair matrix cell (stem cell)


Cells of Wet Stratified Barrier Epithelia


surface epithelial cell of stratified squamous epithelium of cornea,


tongue, oral


cavity, esophagus, anal canal, distal urethra, vagina


basal cell of these epithelia (stem cell)


cell of urinary epithelium (lining bladder and urinary ducts)


Epithelial Cells Specialized for Exocrine Secretion


cells of salivary gland









mucous cell (secretion rich in polysaccharide)



serous cell (secretion rich in glycoprotein enzymes)







cell of von Ebner's gland in tongue (secretion to wash over taste


buds)


cell of mammary gland, secreting milk


cell of lacrimal gland, secreting tears


cell of ceruminous gland of ear, secreting wax


cell of eccrine sweat gland, secreting glycoproteins (dark cell)


cell of eccrine sweat gland, secreting small molecules (clear cell)


cell of apocrine sweat gland (odoriferous secretion, sex-hormone


sensitive)


cell of gland of Moll in eyelid (specialized sweat gland)


cell of sebaceous gland, secreting lipid-rich sebum


cell of Bowman's gland in nose (secretion to wash over olfactory


epithelium)


cell of Brunner's gland in duodenum, secreting alkaline solution of


mucus and enzymes


cell of seminal vesicle, secreting components of seminal fluid,


including fructose (as fuel for swimming sperm)


cell of prostate gland, secreting other components of seminal fluid


cell of bulbourethral gland, secreting mucus


cell of Bartholin's gland, secreting vaginal lubricant


cell of gland of Littre, secreting mucus


cell of endometrium of uterus, secreting mainly carbohydrates


isolated goblet cell of respiratory and digestive tracts, secreting


mucus


mucous cell of lining of stomach


zymogenic cell of gastric gland, secreting pepsinogen


oxyntic cell of gastric gland, secreting HCl


acinar cell of pancreas, secreting digestive enzymes and


bicarbonate


Paneth cell of small intestine, secreting lysozyme


type II pneumocyte of lung, secreting surfactant


Clara cell of lung (function unknown)


Cells Specialized for Secretion of Hormones


cells of anterior pituitary, secreting growth hormone, follicle-


stimulating hormone, luteinizing hormone, prolactin,


adrenocorticotropic hormone, and/or thyroid-stimulating hormone


cell of intermediate pituitary, secreting melanocyte-stimulating


hormone


cells of posterior pitutiary, secreting oxytocin and/or vasopressin


cells of gut and respiratory tract, secreting serotonin, endorphin,


somatostatin, gastrin, secretin, cholecystokinin, insulin, glucagon,


and/or bombesin


cells of thyroid gland, secreting









thyroid hormone



calcitonin







cells of parathyroid gland, secreting









parathyroid hormone



oxyphil cell (function unknown)







cells of adrenal gland, secreting









epinephrine



norepinephrine







steroid hormones









mineralocorticoids



glucocorticoids







cells of gonads, secreting









testosterone (Leydig cell of testis)



estrogen (theca interna cell of ovarian follicle)



progesterone (corpus luteum cell of ruptured ovarian follicle)







cells of juxtaglomerular apparatus of kidney


juxtaglomerular cell (secreting renin)











macula densa cell

(uncertain but probably related in



peripolar cell
{
function; possibly involved in secretion



mesangial cell

of erythropoietin)







Epithelial Absorptive Cells in Gut, Exocrine Glands, and Urogenital Tract


brush border cell of intestine (with microvilli)


striated duct cell of exocrine glands


gall bladder epithelial cell


brush border cell of proximal tubule of kidney


distal tubule cell of kidney


nonciliated cell of ductulus efferens


epididymal principal cell


epididymal basal cell


Cells Specialized for Metabolism and Storage


hepatocyte (liver cell)


fat cells









white fat



brown fat



lipocyte of liver







Epithelial Cells Serving Primarily a Barrier Function, Lining


the Lung, Gut, Exocrine Glands, and Urogenital Tract


type I pneumocyte (lining air space of lung)


pancreatic duct cell (centroacinar cell)


nonstriated duct cell of sweat gland, salivary gland, mammary


gland, etc.









(various)







parietal cell of kidney glomerulus


podocyte of kidney glomerulus


cell of thin segment of loop of Henle (in kidney)


collecting duct cell (in kidney)


duct cell of seminal vesicle, prostate gland, etc. (various)


Epithelial Cells Lining Closed Internal Body Cavities


vascular endothelial cells of blood vessels and lymphatics









fenestrated



continuous



splenic







synovial cell (lining joint cavities, secreting largely hyaluronic


acid)


serosal cell (lining peritoneal, pleural, and pericardial cavities)


squamous cell lining perilymphatic space of ear


cells lining endolymphatic space of ear









squamous cell



columnar cells of endolymphatic sac









with microvilli



without microvilli









“dark” cell



vestibular membrane cell



stria vascularis basal cell



stria vascularis marginal cell



cell of Claudius



cell of Boettcher







choroid plexus cell (secreting cerebrospinal fluid)


squamous cell of pia-arachnoid


cells of ciliary epithelium of eye









pigmented



nonpigmented







corneal “endothelial” cell


Ciliated Cells with Propulsive Function


Ciliated Cells of respiratory tract


Ciliated Cells of oviduct and of endometrium of uterus (in female)


Ciliated Cells of rete testis and ductulus efferens (in male)


Ciliated Cells of central nervous system (ependymal cell lining


brain cavities)


Cells Specialized for Secretion of Extracellular Matrix


epithelial









ameloblast (secreting enamel of tooth)



planum semilunatum cell of vestibular apparatus of ear









(secreting proteoglycan)









interdental cell of organ of Corti (secreting tectorial



“membrane” covering



hair cells of organ of Corti)







nonepithelial (connective tissue)









fibroblasts (various-of loose connective tissue, of cornea, of









tendon, of reticular tissue of bone marrow,



etc.)









pericyte of blood capillary



nucleus pulposus cell of intervertebral disc



cementoblast/cementocyte (secreting bonelike cementum of









root of tooth)









odontoblast/odontocyte (secreting dentin of tooth)



chondrocytes









of hyaline cartilage



of fibrocartilage



of elastic cartilage









osteoblast/osteocyte



osteoprogenitor cell (stem cell of osteoblasts)



hyalocyte of vitreous body of eye



stellate cell of perilymphatic space of ear







Contractile Cells


skeletal muscle cells









red (slow)



white (fast)



intermediate



muscle spindle-nuclear bag



muscle spindle-nuclear chain



satellite cell (stem cell)







heart muscle cells









ordinary



nodal



Purkinje fiber







smooth muscle cells (various)


myoepithelial cells









of iris



of exocrine glands







Cells of Blood and Immune System


red blood cell


megakaryocyte


macrophages and related cells









monocyte



connective-tissue macrophage (various)



Langerhans cell (in epidermis)



osteoclast (in bone)



dendritic cell (in lymphoid tissues)



microglial cell (in central nervous system)







neutrophil


eosinophil


basophil


mast cell


T lymphocyte









helper T cell



suppressor T cell



killer T cell







B lymphocyte









IgM



IgG



IgA



IgE







killer cell


stem cells and committed progenitors for the blood and









immune system (various)







Sensory Transducers


photoreceptors









rod



cones









blue sensitive



green sensitive



red sensitive







hearing









inner hair cell of organ of Corti



outer hair cell of organ of Corti







acceleration and gravity









type I hair cell of vestibular apparatus of ear



type II hair cell of vestibular apparatus of ear







taste









type II taste bud cell







smell









olfactory neuron







basal cell of olfactory epithelium (stem cell for olfactory neurons)


blood pH









carotid body cell









type I



type II







touch









Merkel cell of epidermis



primary sensory neurons specialized for touch (various)







temperature









primary sensory neurons specialized for temperature









cold sensitive



heat sensitive







pain









primary sensory neurons specialized for pain (various)







configurations and forces in musculoskeletal system









proprioceptive primary sensory neurons (various)







Autonomic Neurons


cholinergic (various)


adrenergic (various)


peptidergic (various)


Supporting Cells of Sense Organs and of Peripheral Neurons


supporting cells of organ of Corti


inner pillar cell









outer pillar cell



inner phalangeal cell



outer phalangeal cell



border cell



Hensen cell







supporting cell of vestibular apparatus


supporting cell of taste bud (type I taste bud cell)


supporting cell of olfactory epithelium


Schwann cell


satellite cell (encapsulating peripheral nerve cell bodies)


enteric glial cell


Neurons and Glial Cells of Central Nervous System


neurons (huge variety of types-still poorly classified)


glial cells









astrocyte (various)



oligodendrocyte







Lens Cells


anterior lens epithelial cell


lens fiber (crystallin-containing cell)


Pigment Cells


melanocyte


retinal pigmented epithelial cell


Germ Cells


oogonium/oocyte


spermatocyte


spermatogonium (stem cell for spermatocyte)


Nurse Cells


ovarian follicle cell


Sertoli cell (in testis)


thymus epithelial cell


Exocrine secretory epithelial cells


Salivary gland mucous cell (polysaccharide-rich secretion)


Salivary gland number 1 (glycoprotein enzyme-rich secretion)


Von Ebner's gland cell in tongue (washes taste buds)


Mammary gland cell (milk secretion)


Lacrimal gland cell (tear secretion)


Ceruminous gland cell in ear (earwax secretion)


Eccrine sweat gland dark cell (glycoprotein secretion)


Eccrine sweat gland clear cell (small molecule secretion)


Apocrine sweat gland cell (odoriferous secretion, sex-hormone


sensitive)


Gland of Moll cell in eyelid (specialized sweat gland)


Sebaceous gland cell (lipid-rich sebum secretion)


Bowman's gland cell in nose (washes olfactory epithelium)


Brunner's gland cell in duodenum (enzymes and alkaline


mucus)


Seminal vesicle cell (secretes seminal fluid components,


including fructose for swimming sperm)


Prostate gland cell (secretes seminal fluid components)


Bulbourethral gland cell (mucus secretion)


Bartholin's gland cell (vaginal lubricant secretion)


Gland of Littre cell (mucus secretion)


Uterus endometrium cell (carbohydrate secretion)


Isolated goblet cell of respiratory and digestive tracts (mucus


secretion)


Stomach lining mucous cell (mucus secretion)


Gastric gland zymogenic cell (pepsinogen secretion)


Gastric gland oxyntic cell (hydrochloric acid secretion)


Pancreatic acinar cell (bicarbonate and digestive enzyme


secretion)


Paneth cell of small intestine (lysozyme secretion)


Type II pneumocyte of lung (surfactant secretion)


Clara cell of lung


Hormone secreting cells


Anterior pituitary cells









Somatotropes



Lactotropes



Thyrotropes



Gonadotropes



Corticotropes







Intermediate pituitary cell, secreting melanocyte-stimulating


hormone


Magnocellular neurosecretory cells









secreting oxytocin



secreting vasopressin







Gut and respiratory tract cells









secreting serotonin



secreting endorphin



secreting somatostatin



secreting gastrin



secreting secretin



secreting cholecystokinin



secreting insulin



secreting glucagon



secreting bombesin







Thyroid gland cells









thyroid epithelial cell



parafollicular cell







Parathyroid gland cells









Parathyroid chief cell



Oxyphil cell







Adrenal gland cells









chromaffin cells



secreting steroid









hormones (mineralcorticoids and gluco



corticoids)







Leydig cell of testes secreting testosterone


Theca interna cell of ovarian follicle secreting estrogen


Corpus luteum cell of ruptured ovarian follicle


secreting progesterone









Granulosa lutein cells



Theca lutein cells







Juxtaglomerular cell (renin secretion)


Macula densa cell of kidney


Peripolar cell of kidney


Mesangial cell of kidney


Derived primarily from ectoderm


Integumentary system


Keratinizing epithelial cells


Epidermal keratinocyte (differentiating epidermal cell)


Epidermal basal cell (stem cell)


Keratinocyte of fingernails and toenails


Nail bed basal cell (stem cell)


Medullary hair shaft cell


Cortical hair shaft cell


Cuticular hair shaft cell


Cuticular hair root sheath cell


Hair root sheath cell of Huxley's layer


Hair root sheath cell of Henle's layer


External hair root sheath cell


Hair matrix cell (stem cell)


Wet stratified barrier epithelial cells


Surface epithelial cell of stratified squamous


epithelium of cornea, tongue, oral cavity, esophagus, anal


canal, distalurethra and vagina


basal cell (stem cell) of epithelia of cornea, tongue, oral cavity,


esophagus, anal canal, distal urethra and vagina


Urinary epithelium cell (lining urinary bladder and urinary


ducts)


Nervous system


There are nerve cells, also known as neurons, present in our human


body. They are branched out. These cells make upnervous tissue.


A neuron consists of a cell body with a nucleus and cytoplasm,


from which long thin hair-like parts arise.


Sensory transducer cells


Auditory inner hair cell of organ of Corti


Auditory outer hair cell of organ of Corti


Basal cell of olfactory epithelium (stem cell for olfactory


neurons)


Cold-sensitive primary sensory neurons


Heat-sensitive primary sensory neurons


Merkel cell of epidermis (touch sensor)


Olfactory receptor neuron


Pain-sensitive primary sensory neurons (various types)


Photoreceptor cells of retina in eye:









Photoreceptor rod cells



Photoreceptor blue-sensitive cone cell of eye



Photoreceptor green-sensitive cone cell of eye



Photoreceptor red-sensitive cone cell of eye







Proprioceptive primary sensory neurons (various types)


Touch-sensitive primary sensory neurons (various types)


Type I carotid body cell (blood pH sensor)


Type II carotid body cell (blood pH sensor)


Type I hair cell of vestibular system of ear (acceleration and


gravity)


Type II hair cell of vestibular system of ear (acceleration and


gravity)


Type I taste bud cell


Autonomic neuron cells


Cholinergic neural cell


Adrenergic neural cell


Peptidergic neural cell


Sense organ and peripheral neuron supporting cells


Inner pillar cell of organ of Corti


Outer pillar cell of organ of Corti


Inner phalangeal cell of organ of Corti


Outer phalangeal cell of organ of Corti


Border cell of organ of Corti


Hensen cell of organ of Corti


Vestibular apparatus supporting cell


Taste bud supporting cell


Olfactory epithelium supporting cell


Schwann cell


Satellite glial cell (encapsulating peripheral nerve cell bodies)


Enteric glial cell


Central nervous system neurons and glial cells


Astrocyte (various types)


Neuron cells (large variety of types, still poorly classified)


Oligodendrocyte


Spindle neuron


Lens cells


Anterior lens epithelial cell


Crystallin-containing lens fiber cell


Derived primarily from mesoderm


Metabolism and storage cells


Hepatocyte (liver cell)


Adipocytes:









White fat cell



Brown fat cell







Liver lipocyte


Barrier function cells (lung, gut, exocrine


glands and urogenital tract)


Kidney


Kidney parietal cell


Kidney glomerulus podocyte


Kidney proximal tubule brush border cell


Loop of Henle thin segment cell


Kidney distal tubule cell


Kidney collecting duct cell[disambiguation needed]


Type I pneumocyte (lining air space of lung cell)


Pancreatic duct cell (centroacinar cell)


Nonstriated duct cell (of sweat gland, salivary gland, mammary


gland, etc.)









principal cell



Intercalated cell







Duct cell (of seminal vesicle, prostate gland, etc.)


Intestinal brush border cell (with microvilli)


Exocrine gland striated duct cell


Gall bladder epithelial cell


Ductulus efferens nonciliated cell


Epididymal principal cell


Epididymal basal cell


Extracellular matrix cells


Ameloblast epithelial cell (tooth enamel secretion)


Planum semilunatum epithelial cell of vestibular system of ear


(proteoglycan secretion)


Organ of Corti interdental epithelial cell (secreting tectorial


membrane covering hair cells)


Loose connective tissue fibroblasts


Corneal fibroblasts (corneal keratocytes)


Tendon fibroblasts


Bone marrow reticular tissue fibroblasts


Other nonepithelial fibroblasts


Pericyte


Nucleus pulposus cell of intervertebral disc


Cementoblast/cementocyte (tooth root bonelike ewan cell


secretion)


Odontoblast/odontocyte (tooth dentin secretion)


Hyaline cartilage chondrocyte


Fibrocartilage chondrocyte


Elastic cartilage chondrocyte


Osteoblast/osteocyte


Osteoprogenitor cell (stem cell of osteoblasts)


Hyalocyte of vitreous body of eye


Stellate cell of perilymphatic space of ear


Hepatic stellate cell (Ito cell)


Pancreatic stelle cell


Contractile cells


skeletal muscle Cell









Red skeletal muscle cell (slow)



White skeletal muscle cell (fast)



Intermediate skeletal muscle cell



nuclear bag cell of muscle spindle



nuclear chain cell of muscle spindle







Satellite cell (stem cell)


Heart muscle cells









Ordinary heart muscle cell



Nodal heart muscle cell



Purkinje fiber cell







Smooth muscle cell (various types)


Myoepithelial cell of iris


Myoepithelial cell of exocrine glands


Blood and immune system cells


Erythrocyte (red blood cell)


Megakaryocyte (platelet pecursor)


Monocyte (white blood cell)


Connective tissue macrophage (various types)


Epidermal Langerhans cell


Osteoclast (in bone)


Dendritic cell (in lymphoid tissues)


Microglial cell (in central nervous system)


Neutrophil granulocyte


Eosinophil granulocyte


Basophil granulocyte


Hybridoma cell


Mast cell


Helper T cell


Suppressor T cell


Cytotoxic T cell


Natural Killer T cell


B cell


Natural killer cell


Reticulocyte


Stem cells and committed progenitors for the blood and immune


system (various types)


Germ cells


Oogonium/Oocyte


Spermatid


Spermatocyte


Spermatogonium cell (stem cell for spermatocyte)


Spermatozoon


Nurse cells


Ovarian follicle cell


Sertoli cell (in testis)


Thymus epithelial cell


Interstitial cells


Interstitial kidney cells
















TABLE 7







B Cell maturation markers for use with the


hydrogel particles described herein.








B-cell



type
Cell surface marker(s)





Pro-B
CD19, CD20, CD34, CD38, CD45R


Pre-B
CD19, CD20, CD38, CD45R


Immature B
CD19, CD20, CD40, CD45R, IgM


Tr-B
CD10, CD19, CD20, CD24, CD28


Naïve-B
CD19, CD20, CD23, CD40, CD150 (SLAM), IgD, IgM


B-1
CD19, CD20, CD27, IgM


Memory B
CD19, CD20, CD28, CD40, IgA, IgG


Plasma Cell
CD19, CD28, CD31, CD38, CD40, CD95 (FAS), CD184



(CXCR4)
















TABLE 8





Cell surface markers for use with the


hydrogel particles described herein.

















14-3-3 Î ± Î2



14-3-3 Îμ



14-3-3 Î¶



14-3-3 Î,



14-3-3 {hacek over (I)}f



15-Lipoxygenase 1



160 kD Neurofilament Medium



200 kD Neurofilament Heavy



2H2



3G11 sialoganglioside antigen



4E-BP1



4E-BP1 Phospho (Thr37/46)



5-Methylcytidine



5HT3A receptor



5T4



68 kDa Neurofilament Light



7.1



70 kD Neurofilament Light



A20



A2B5



AAK1



ABCA1



ABCA7



ABCB4



ABCB5



ABCC10



ABCC11



ABCG1



ABI2



ABIN3



ABIN3Î2



ABL2



Abraxas



ACAA1



ACADM



ACAT2



ACBD3



ACD



ACE2



Acetyl Coenzyme A Carboxylase



Acetyl Coenzyme A Carboxylase Î±



Acetyl Coenzyme A Synthetase



Acetylated Lysine



AChRÎ±



AChRÎ2



AChRÎ3



Aconitase2



ACOT12



ACSA2



ACSF2



ACSM5



Act1



Activation molecule 8 (B cells)



Activin A Receptor Type IB



Activin A Receptor Type IIB



ACTN3



ACY1



ACY3



ADA



ADAM12



ADE2



Adenosine A1 Receptor



Adenosine A2aR



Adenovirus



Adenovirus Fiber monomer and trimer



Adenovirus hexon protein



Adenylate Kinase 1



Adenylosuccinate Lyase



ADFP



ADH1B



ADH6



ADH7



ADI1



Adiponectin



Adiponectin Receptor 2



Adipose Triglyceride Lipase



ADP Ribosylation Factor



ADP-ribosyltransferase 2.2 gene



Adrenodoxin



AF10



AFAP1



AFP



AG2



AGAP1



AGPAT5



AGR2



AHSG



AICDA



AID



AIF



AIM-2



Aiolos



AIPL1



AIRE



AK3



AK3L1



AK5



Akt



Akt (pS473)



Akt (pT308)



Akt1



Akt2



Akt3



Albumin



Alcohol Dehydrogenase



Aldehyde Reductase



ALDH1A1



ALDH1L1



ALDH2



ALDH3A1



ALDH3A2



ALDH5A1



ALDH6A1



ALDH7A1



ALDOB



Aldolase B



Alexa Fluor 405/Cascade Blue



Alexa Fluor 488



ALG2



Alix



Allergin1



alpha 1 Antitrypsin



alpha 1 Catenin



alpha 1 Sodium Potassium ATPase



alpha 2 Catenin



alpha 2 Macroglobulin



alpha Actin 1



alpha Actin 2



alpha Actinin



alpha Actinin 2



alpha Actinin 3



alpha Actinin 4



alpha Adaptin



alpha Adducin



alpha B Crystallin



alpha Fodrin



alpha Internexin



alpha Synuclein



ALS1



AMACR



Aminopeptidase P



AML1



Amphiphysin



AMPKÎ±



AMPKÎ ± 1



AMPKÎ ± 2



AMPKÎ21



AMPKÎ31



AmyloidÎ2 42



ANAPC2



AND1



Androgen Receptor



Angiotensin I



Angiotensin II Receptor 2



Angiotensin III



ANKRD53



Annexin IV



Annexin V



ANP



Anti-Kudoa thrysites



Anti-T. brucei procyclin (GPEET)



Anti-T. brucei procyclin (phosphorylated GPEET)



Antiglobulin (Coombs)



Antithrombin III



AP2 Î±



AP2 Î ± Î2



AP2 Î3



AP2M1



AP2S1



APAF1



APBB3



APC



APC-1



APC-10



APC-11



APC-2



APC-3



APC-5



APC-7



APC-8



APE1



APG12



APG3



APG5



APG7



APMAP



Apo-2.7



Apo-2.7 (7A6)



ApoE



ApoE4



APOER2



Apolipoprotein AI



Apolipoprotein AII



Apolipoprotein AIV



Apolipoprotein B



Apolipoprotein CIII



Apolipoprotein D



Apolipoprotein E



Apolipoprotein F



Apolipoprotein H



Apolipoprotein J



Apolipoprotein L1



Apolipoprotein M



Apoptotic neutrophils



APP



Aquaporin 1



Aquaporin 5



ARF1



ARF5



ARFGAP1



ARFRP1



Argonaute-1



ARH



ARHGAP25



ARHGAP4



ARL11



ARL5B



ARPC5



Artemis



Aryl hydrocarbon Receptor



ASB-1



ASCC1



ASCC2



ASGPR



Asialo-GM1



ASK1



Asparagine synthetase



Ataxin 1



ATF1



ATF2



ATG4A



ATG9A



ATIC



Atlantic Salmon Ig



ATM



ATP citrate lyase



ATP1B3



ATP5A



ATP5H



ATP5J



ATP5O



ATP6V0D1



ATP6V1B1



ATPB



ATRIP



Aurora A



Aurora A Phospho (Thr288)



Aurora B



Aurora B Phospho (Thr232)



AVEN



Avian Influenza A Neuraminidase



Avidin



Axin 2



Axl



B and Activated T Cells



B Cell



B Cell Subset



B cells (pan reactive)



B lymphocytes antibody [UCH-B1]



b-Endorphin



B-Raf Phospho (Thr598/Ser601)



B18R



B7-H4



BACE1



BACE2



BACH1



baculovirus envelope gp64 protein



BAG1



BAG2



BAG3



BAG4



BAIAP2



BAK



BAMBI



BAP31



BAP37



basal cell Cytokeratin



Basophils



Bassoon



BATF



Bax



BCAR1



BCAR2



BCKD complex E2 subunit



Bcl-10



Bcl-2



Bcl-2 (pS70)



Bcl-2 like 12



Bcl-2 like 2



Bcl-22



Bcl-2A1



Bcl-2Î±



Bcl-3



Bcl-6



Bcl-xL



Bcl-XS/L



BCR



BCSC1



BDH2



BDKRB2



BDNF



Beclin1



Bestrophin 3



beta 2 Adrenoreceptor



Beta 3 Adrenergic Receptor



beta 3 Sodium Potassium ATPase



beta Actin



beta Arrestin 1



beta Arrestin 2



beta Catenin



beta Catenin (npaa 27-37)



beta Catenin (npaa 35-50)



beta Catenin (pS45)



beta Dystroglycan



beta galactosidase



beta galactosidase fusion proteins



beta Synuclein



beta2 Microglobulin



BHMT



Bid



Biglycan



Bilirubin Oxidase



Bim



BimL



BIN1



BIN3



Biotin



BiP



BLBP



Blimp-1



BLK



BLNK



BLNK (pY84)



Blood Group A Antigen



Blood Group AB Antigen



Blood Group B Antigen



Blood Group H ab Antigen



Blood Group H ab Antigen/n Antigen



Blood Group H inhibitor



Blood Group Lewis a



Blood Group M Antigen



Blood Group N Antigen



Blooms Syndrome Protein Blm



BM1



BMAL1



BMI1



Bmk



BMP15



BMP4



BMP7



BMPR1A



BMPR2



BMX



bMyc



BNIP2



BNIP3



BNIP3L



BOB1



BORA



Borealin



Borrelia burgdorferi



BPI



BRaf



BRCA1



BRCC36



BRD3



BrdU



BRF1



BRG1



BRN3A



Btk



Btk (pY551)/Itk (pY511)



BTLN-2



BTN1A1



Bu1



Bu1a



Bu1a/Bu1b



Bu1b



BubR1



Bulb



Butyrylcholinesterase



C peptide



C reactive protein



C/EBPÎ2



C1 Inhibitor



C15orf40



C16orf72



C1orf50



C1Q



C1QA



C1QB



C1QC



C1QG



C1r



C1s



C20orf30



C20orf43



C21orf56



C21orf59



C2orf43



C3



C3aR



C3b



C3c



C3d



C4



C4 binding protein



C4b



C4c



C4d



C4orf42



C5



C5aR1



C5L2



C6



C6orf64



C8A/B/G



C9



C9orf41



CA125



CA19.9



CAB39



CACNA1S



CACNA2



CACNG1



CAD



Cadherin 1



Cadherin 10



Cadherin 11



Cadherin 7



Cadherin 8



Cadherin 9



Cadherin E



Cadherin H



Cadherin K



Cadherin P



Cadherin R



CAK C Terminus



CAK N Terminus



CAK Phospho (Ser164/Thr170)



Calbindin



Calcineurin A



Calcitonin Receptor



Calcium Sensing Receptor



Caldesmon



Calgranulin A



Calgranulin B



Calmodulin



Calnexin - ER membrane marker



Calpain 1



Calpain 2



Calpain 9



Calpain S1 (small subunit)



Calpastatin



Calponin



Calreticulin



Calretinin



Calsequestrin 2



CaMKI



CaMKII



CaMKII Phospho (Thr286)



CaMKIIÎ′



CamKIV



CaMKIÎ±



CAMLG



cAMP Protein Kinase Catalytic subunit



cAMP Protein Kinase Catalytic subunit Î±



Cannabinoid Receptor I



Cannabinoid Receptor II



CAP-G2



CAP18



CAP2



CAP3



Carbonic Anhydrase I



Carbonic Anhydrase IX



Carboxylesterase 1



Carboxypeptidase A1



Carboxypeptidase A2



CARD11



CARD8



CARD9



Cardiac Troponin T



CARKL



CARM1



Casein Kinase 1 Î±



Casein Kinase 1 Î32



Casein Kinase 2 Î2



Caspase 1



Caspase 10



Caspase 11



Caspase 12



Caspase 2



Caspase 2L



Caspase 3



Caspase 4



Caspase 5



Caspase 6



Caspase 7



Caspase 8



Caspase 9



Catalase



Catechol-O-methyltransferase



Cathepsin D



Cathepsin K



Cathepsin L



Caveolin1



Caveolin1 (pY14)



Caveolin2



Cbl



CBP



CBWD1



CBX1



cCbl (pY700)



cCbl (pY774)



CCDC98



CCK4



CCL11



CCL17



CCL18



CCL19-Fc



CCL20



CCL21



CCL25



CCL3



CCL5



CCL6



CCNB1IP1



CCR10



CCR11



CCRD6



CCRL2



CD1



CD1.1



CD10



CD100



CD101



CD102



CD103



CD104



CD105



CD106



CD107a



CD107b



CD108



CD109



CD11



CD110



CD111



CD112



CD113



CD114



CD115



CD116



CD117



CD118



CD119



CD11a



CD11a, strain polymorphism



CD11a/CD18



CD11b



CD11b/c



CD11c



CD11d



CD120a



CD120b



CD121a



CD121b



CD122



CD123



CD124



CD125



CD126



CD127



CD129



CD13



CD130



CD131



CD132



CD133



CD133/2



CD134



CD135



CD136



CD137



CD137L



CD138



CD139



CD14



CD140a



CD140b



CD140b (pY1009)



CD140b (pY1021)



CD140b (pY771)



CD140b (pY857)



CD141



CD142



CD143



CD144



CD146



CD147



CD148



CD15



CD150



CD151



CD152



CD153



CD154



CD155



CD156c



CD157



CD158a



CD158a/h



CD158b



CD158b1/b2/j



CD158d



CD158e



CD158e/k



CD158e1



CD158e1/e2



CD158f



CD158g



CD158h



CD158i



CD158j



CD159a



CD159c



CD15s



CD16



CD16/32



CD16/56



CD160



CD161



CD161a



CD162



CD162R



CD163



CD164



CD165



CD166



CD167a



CD168



CD169



CD16b



CD17



CD170



CD171



CD172



CD172a



CD172a/b



CD172b



CD172g



CD173



CD177



CD178



CD178.1



CD179a



CD179b



CD18



CD180



CD181



CD182



CD183



CD184



CD185



CD186



CD19



CD191



CD192



CD193



CD194



CD195



CD195 (cytoplasmic)



CD195 Phospho (Ser337)



CD195 Phospho (Ser349)



CD196



CD197



CD198



CD199



CD1a



CD1b



CD1b/c



CD1c



CD1d



CD1d Î ± GalCer Complex



CD2



CD20



CD200



CD200R



CD200R3



CD201



CD202b



CD203a



CD203c



CD204



CD205



CD206



CD207



CD208



CD209



CD209b



CD21



CD21/CD35



CD210



CD212



CD213a1



CD213a2



CD217



CD218a



CD22



CD22 (pY822)



CD22.2



CD220



CD220Î±



CD221



CD221 (pY1131)



CD222



CD223



CD224



CD226



CD227



CD229



CD229.1



CD23



CD230



CD231



CD233



CD234



CD235a



CD235ab



CD236



CD239



CD24



CD240CE



CD240DCE



CD243



CD244



CD244.1



CD244.2



CD245



CD246



CD247



CD247 (pY142)



CD249



CD25



CD252



CD253



CD254



CD255



CD256



CD257



CD258



CD26



CD261



CD262



CD263



CD264



CD265



CD266



CD267



CD268



CD269



CD27



CD270



CD271



CD272



CD273



CD274



CD275



CD276



CD277



CD278



CD279



CD28



CD280



CD281



CD282



CD283



CD284



CD284/MD2 Complex



CD286



CD289



CD29



CD290



CD294



CD298



CD299



CD2a



CD3



CD3/CD44



CD30



CD300



CD300a



CD300e



CD300f



CD301



CD303



CD303a



CD304



CD305



CD307d



CD309



CD31



CD310



CD312



CD314



CD314 (activating)



CD314 (blocking)



CD317



CD318



CD319



CD32



CD321



CD323



CD324



CD325



CD326



CD328



CD329



CD32B



CD33



CD334



CD335



CD336



CD337



CD338



CD339



CD34



CD340



CD344



CD349



CD35



CD351



CD354



CD357



CD358



CD36



CD360



CD361



CD36L1



CD37



CD38



CD39



CD39L4



CD3D



CD3G



CD3Î3



CD3Î′



CD3Îμ



CD3Îμ (CD3 Molecular Complex)



CD4



CD4 (domain 1)



CD4 (domain 2)



CD4 v4



CD40



CD40bp



CD41



CD41/CD61



CD41a



CD41b



CD42a



CD42b



CD42d



CD43



CD44



CD44 (v3)



CD44 (v4)



CD44 (v5)



CD44 (v6)



CD44 (v7)



CD44.2



CD44std



CD44v6



CD44var (v10)



CD44var (v3)



CD44var (v3-v10)



CD44var (v4)



CD44var (v5)



CD44var (v6)



CD44var (v7)



CD44var (v7-v8)



CD45



CD45.1



CD45.2



CD45R



CD45RA



CD45RB



CD45RC



CD45RO



CD46



CD47



CD48



CD49a



CD49a/CD29



CD49b



CD49b/CD29



CD49b/CD61



CD49c



CD49d



CD49d/CD29



CD49e



CD49e/CD29



CD49f



CD49f/CD29



CD4Î±



CD5



CD5.1



CD5.2



CD5.6



CD50



CD51



CD51/61



CD52



CD53



CD54



CD55



CD56



CD57



CD58



CD59



CD59a



CD6



CD60b



CD61



CD62E



CD62L



CD62P



CD63



CD64



CD64 a, b alloantigens



CD64.1



CD65



CD65s (CD65 sialylated)



CD66



CD66a



CD66a/b/c/e



CD66a/c/d



CD66a/c/d/e



CD66a/c/e



CD66a/e



CD66b



CD66c



CD66c/e



CD66e



CD66f



CD68



CD69



CD7



CD70



CD70b



CD71



CD72



CD72 a, b, c alloantigens



CD72 b, c alloantigens



CD72.1



CD73



CD74



CD75



CD77



CD78



CD79a



CD79b



CD8



CD80



CD81



CD82



CD83



CD84



CD85



CD85a



CD85d



CD85g



CD85h



CD85j



CD85k



CD86



CD87



CD88



CD89



CD8Î±



CD8Î ± .1



CD8Î ± .2



CD8Î2



CD9



CD90.1



CD90.2



CD90.9



CD91



CD91Î±



CD91Î2



CD93



CD94



CD95



CD96



CD97



CD98



CD98hc



CD99



CD99R



Cdc-123



Cdc-2 (p34)



Cdc-25A Phosph (Ser17)



Cdc-25C



Cdc-37



Cdc-45L



Cdc-6



CDc-7



Cdk1



Cdk2



Cdk4



Cdk5



Cdk6



Cdk7



Cdk9



CdkA1



CdkN2A



CdkN3



CDT1



CDX2



CEACAM19



CEACAM20



CEACAM7



CEBPÎ±



CEBPÎ2



CEND1



CENPA



CENPE



CENPF



CENPH



Centrin 2



CFAH



cFos



CFTR



CGB5



cGK1



CH2



CHCHD5



CHD3



CHD4



Chemerin



CHIPS, C-terminus



CHIPS, N-terminus



Chk1



Chk2



Chondroitin Sulfate



CHOP



Chromogranin C



ChT1



chTOG



cIAP1



cIAP2



CIAS1



CIDEA



CIP4



CISD1



CITED1



CITED2



cJun



cJun Phospho (Tyr91/Tyr93)



CKIIÎ±



CKMT2



CLASP1



Clathrin



Claudin-1



Claudin-10



Claudin-15



Claudin-16



Claudin-18 (C-term)



Claudin-18 (Mid)



Claudin-4



Claudin-5



Claudin-8



CLAW-H



CLEC12A



CLEC1B



CLEC4A



CLEC4M



CLEC9A



CLIP



CLOCK



Clostridium botulinum Toxin B



CLPP



cMaf



cMet



CMKLR1



CMRF44



CMRF56



cMyb



cMyc



CNDP2



CNTFRÎ±



COASY



Coatomer Î′



Cofilin



Colec12



Collagen I



Collagen I/III



Collagen II



Collagen III



Collagen IV



Collagen V



Collagen VI



Collagen VII



COMMD1



Complement Factor B



Complex I Immunocapture



Conjugated Choline Glutaric acid



Connexin 26



Connexin 30



Connexin 30.2



Connexin 30.3



Connexin 32



Connexin 36



Connexin 37



Connexin 37 (C-term)



Connexin 37 (Mid)



Connexin 39



Connexin 39 (Mid)



Connexin 40 (C-term)



Connexin 40 (Mid)



Connexin 43



Connexin 45



Connexin 45 (C-term)



Connexin 46



Connexin 47



Connexin 57 (C-term)



Connexin 57 (Mid)



Contactin 2



COPS3



Coronavirus



Coronin 1A



Coronin 1B



Cortactin



Cortical Thymocytes



COX I



COX I/III



COX II



COX IV



COX VA



COX VIA1



Coxsackie Adenovirus Receptor



CPF



CPI17Î±



Cpn10



CPO



CPS1



CPT2



CRABP1



CRABP2



CRALBP



Creatine Kinase BB



Creatine Kinase MM



CREB



CREB Phospho (Ser133)



cRel



Cripto1



CRISP3



Crk p38



CrkL



CrkL (pY207)



CROT



CRRY



CRTAM



CRTC3



CRY2



Cryptochrome I



Cryptosporidium



Cryptosporidium Parvum



CRYZL1



CSK



CSK Binding Protein



CSPS



cSrc



CST2



CTDSP1



CTNNA3



CTNNBL1



Cullin 1



Cullin 2



Cullin 3



Cullin 4A



Cullin 4A/B



Cullin 4B



Cutaneous Lymphocyte Antigen



CUTL1



CX3CL1



CX3CR1



CXCL1



CXCL10



CXCL12Î±



CXCL12Î2



CXCL13



CXCL9



CXCR7



CXorf26



Cyanine



CYB5R2



CYB5R3



Cyclin A



Cyclin A2



Cyclin B1



Cyclin B2



Cyclin D1



Cyclin D2



Cyclin D3



Cyclin E



Cyclin E2



Cyclin H



Cyclins D1/D2/D3



Cyclophilin 40



CYLD



CysLT1



Cystatin C



Cystatin S



Cytochrome B245 heavy chain



Cytochrome B245 light chain



Cytochrome c



Cytochrome P450 17A1



Cytochrome P450 19A1



Cytochrome P450 1A2



Cytochrome P450 2A6



Cytochrome P450 2B6



Cytochrome P450 2C9



Cytochrome P450 2J2



Cytochrome P450 3A4



Cytochrome P450 3A5



Cytochrome P450 Reductase



Cytokeratin



Cytokeratin (acidic)



Cytokeratin (basic)



Cytokeratin (Pan-reactive)



Cytokeratin 1



Cytokeratin 10



Cytokeratin 10/13



Cytokeratin 13



Cytokeratin 14



Cytokeratin 14/15/16/19



Cytokeratin 15



Cytokeratin 16



Cytokeratin 17



Cytokeratin 18



Cytokeratin 19



Cytokeratin 2



Cytokeratin 20



Cytokeratin 4



Cytokeratin 4/5/6/8/10/13/18



Cytokeratin 40



Cytokeratin 5



Cytokeratin 5/6/18



Cytokeratin 5/8



Cytokeratin 6



Cytokeratin 6a



Cytokeratin 7



Cytokeratin 7/17



Cytokeratin 8



Cytokeratin 8/18/19



D4-GDI



DAB2



DACH1



DAND5



DAP1



DAP12



DAPK1



DAPK2



DARPP32



Daxx



DAZL



DBC1



DCAMKL1



DCC



DCIR2



DCLRE1B



DCP1a



DcR3



DCTN2



DcTRAIL-R1



DcTRAIL-R2



DCXR



DDB1



DDDDK tag



DDX3



DDX4



DDX50



DECR1



Dectin1



Dectin2



DEF8



Defensin Î ± 1



DELETE



delta 1 Catenin



Delta like protein 1



Delta like protein 4



Delta Opioid Receptor



DeltaC



DeltaD



Dendritic Cell Marker



Deoxycytidine kinase



Desmin



Desmoglein 2



Desmoglein1



Desmoplakin



Destrin



Dextran



DGKA



Dicer



DISC1 (C-term)



DISC1 (Mid)



Dishevelled 3



Disialoganglioside GD2



Disialoganglioside GD3



Dkk1



Dkk3



DLC8



DLK1



Dlx5



DM-GRASP



DMT1



DNA-PKcs



DNA-PKcs Phospho (Thr2609)



DNAI1



DNAJA2



DNAJB2



DNAJC3



DNAPK



DNM1L



Dnmt1



Dnmt3b



DNP



DOK2



DOK7



Dopamine Receptor D1



Dopamine Receptor D3



Dopamine Receptor D5



Dopamine Î2 Hydroxylase



Doublecortin



DP1



DPH2



DPP10



DPP3



DPP9



Dppa4



DPYD



DR3



DRAK1



DRAK2



Drebrin



DTYMK



DUSP23



DUSP27



DUSP3



DUSP5



DUSP6



DUX4



DYKDDDDK Epitope Tag



Dynamin



Dynamin1



Dynamitin



Dynein light chain 2



Dysbindin



Dysferlin



Dystrobrevin Î±



Dystrobrevin Î2



Dystroglycan Phospho (Tyr893)



E. Coli O/E



E2A-Pbx1



E2F1



E47



E4BP4



Ea52-68 peptide bound to I-A



Ea52-68 peptide bound to the I-A



EAAT1



Early B Lineage



EBF1



EBI3



EBP50



ECGF1



ECH1



ECRG4



EDA



EDA-A2R



EDG1



EDG2



EDG3



EDG6



EEA1



EEF1G



EEF2



EEF2K



EEN



EFEMP1



EFEMP2



Eg5



Eg5 Phospho (Thr927)



EGF



EGF Receptor



EGF Receptor (pY1173)



EGF Receptor (pY845)



EGF Receptor (pY992)



EGR1



EGR2



EHD1



eIF1



eIF2C2



EIF2S1



eIF2Î3



eIF3



eIF3D



eIF3D (p66)



eIF3F



eIF3G



eIF3H (p40)



eIF3I (p36)



eIF3J



eIF3K



eIF4B



eiF4E



eIF4E (pS209)



eIF4E2



eIF5A



eIF6



Elastase



Elk1



Elk1 (pS383)



ELK3



Elongin B



Elongin C



EMAP II



Embigin



EMG1



Emi1



EMR3



EMSY



Ena/Vasp-like



EndoG



EndoGlyx-1



Endomucin



Endothelial Cells



Endothelial Lipase



Endothelial Venule Marker



Endothelium



Engrailed1



ENO1



Enolase1



eNOS



eNOS (pS1177)



Entpd2



Eomes



Eos



Epac1



Eph Receptor A1



Eph Receptor A2



Eph Receptor A4



Eph Receptor B4



Eph Receptor B6



Ephrin A2



Ephrin A3



EPHX2



EPM2AIP1



EPOR



EPS15R



Epsin 1



Epsin 2



ER-HR3



ER-MP54



ER-TR7



ER81



ERAB



ERCC1



ERG



ERK1



ERK1/2 (pT185/pY187)



ERK1/2 (pT202/pY204)



ERK1/ERK2



ERK2



ERK5



ERMAP



ERp29



ERp72



Erythroid Cells



Erzin/Radixin/Moesin



ERÎ ± Phospho(Ser167)



ESAM



Estrogen Inducible Protein pS2



Estrogen Receptor



Estrogen Receptor Î±



Estrogen Receptor Î2



Estrogen Related Receptor alpha



ETAR



Ethenoadenosine



ETS1



EVI2A



EVI2B



EWSR1



EXD1



EXOSC3



EXOSC7



EYA2



EZH1/2



Ezrin



Ezrin (pY353)



F-actin



F10A1



F4/80



FAA4



FABP4



Factor I



Factor IX



Factor VIII.vWF (delete)



Factor XIIIa



FADD



FAHD2A



FAK



FAK (pS910)



FAM119A



FAM175A



FAM84B



FAM91A1



FANCC



FANCD2



Fanconi anemia D2 Phospho (Ser222)



FAP



Fascin



FBP1



FBXO21



FBXO31



FBXO42



FBXO43



Fc Receptor Binding Inhibitor



Fc receptor IgA + IgM



FcR



FcRL6



FcRLA



FcÎμRI



FDC



FDFT1



FDPS



FE65



FeLV p27



FEN1



FER



Ferritin Heavy Chain



Ferritin Light Chain



Ferritin, mitochondrial



FES



Fetal Hemoglobin



FGF acidic



FGF basic



FGF21



FGFR1



FGFR2



FGR



FH



FHL1



Fibrillarin



Fibrillin



Fibrinogen



Fibrinogen Î ± chain



Fibrinogen Î3 chain



Fibrinopeptide A



Fibrinopeptide B



Fibroblast activation protein Î±



Fibroblast Surface Protein



Fibroblasts/Epithelial cells



Fibronectin



Fibronectin Receptor



Fibulin5



Ficolin B



Filaggrin



Filamin A



FITC



FITC/Oregon Green



FIV



FIV gp120



FIV gp95



FIV p24



FIV p24 gag



FKBP12



FKBP4



FKBP6



FKBPL



FLiC



Flightless1



FLIP



Flt3L



Fluorescent Protein



FLV gp70



FLYWCH2



FMC7



fMLP Receptor



FMRP



FNTA



FNTB



Follicular Dendritic Cells



Fos



FOXA1



FOXA2



FOXC2



FOXD3



FOXI1



FOXJ1



FOXM1



FOXO1



FOXO3A



FOXP1



FOXP3



FPRL1



FR4



Fra2



Fragilis



FRAT1



Frataxin



Frequenin



Frizzled-1



FSHÎ±



FSHÎ2



FUK



FUS



FXYD3



FYB



Fyn



Fyn (pY528)/c-Src (pY530)



Fyn-Related Kinase



FZR1



G-CSF



G3BP



G6PD



GAB1



GAB2



GABA B Receptor 2



GABARAP



GAD65



GAD67



GADD34



Galacto-cerebroside



Galactocerebroside



Galectin 1



Galectin 10



Galectin 3



Galectin 4



Galectin 7



Galectin 8



Galectin 9



gamma Synuclein



Ganglioside GD2



Ganglioside GD3



Ganglioside GM1



Gankyrin



GAP



GAP43



GAPDH



GARP



GAS2



GAS7



GAT2



GATA1



GATA2



GATA3



GATA4



GATM



GBA3



GBE1



GBP1



GBP2



GBP5



GC1qR



GCDFP15



GCDH



GCK1



GCLM



GCN2



GCN5



GCTM2



GDAP1L1



GDF15



Gelsolin



Gemin1



Gephyrin



GFAP



GFP



GILZ



GIMAP4



GIPR



GIT2



GITRL



GLAST



Gli1



Glial Fibrilary Acidic Protein



Glicentin



GLIPR1L1



Glucagon



Glucocorticoid Receptor



Glucocorticoid Receptor alpha



Glucose 1 Dehydrogenase



Glucose 6 Phosphate Isomerase



GLUH1



GLUT1



GLUT2



GLUT4



GLUT5



Glutamate receptor 2



Glutamate receptor 2/3



Glutamate receptor 3



Glutamate receptor 4



Glutaminase



Glutamine Synthetase



Glutaredoxin 2



Glutathione NEM



Glutathione NEW



Glutathione Peroxidase 1



Glutathione Peroxidase 4



Glutathione Reductase



Glutathione S Transferase Î, 2



Glutathione S Transferase Î°1



Glutathione S Transferase Î1⁄4



Glutathione Synthetase



Glycogen synthase 1



Glycoprotein IX



Glycoprotein VI



GM-CSF



GM130



GM3.2



GNB2



GNB2L1



GNLY



GNMT



GnRHR



Golgi Protein (58K)



Golgi Zone



GOLM1



GOLPH2



GOSR1



gp340



gp49R



GPA33



GPCR5C



GPR-120



GPR-143



GPR-151



GPR-18



GPR-30



GPR-40



GPR-48



GPR-49



GPR-50



GPR-56



GPR-73A



GPR-73B



GPR-77



GPR-83



GPR-86



GPR-C5C



GPR-C5D



Granulin



Granulysin



Granzyme A



Granzyme B



Granzyme K



GRAP2



GRASP1



GRASP65



GRB2



GRB7



GRHPR



GRIM19



GRK1



GRK2



GRK3



GRK5



GRK6



Growth hormone receptor



GRP170



GRP94



GSC



GSK3Î±



GSK3Î±/Î2



GSK3Î2



GSPT2



GST



GST Epitope Tag



GSTA4



GTF2D1



GTPase HRAS



GTPBP4



Guanylate kinase



H-2



H-2.m31



H-2Db



H-2Dd



H-2Kd



H2-M



H2-M3



H2A.X



H2A.X Phospho (Ser139)



H2A1J



H60



HA tag



HADHA



HADHA/HADHB



HADHB



HADHSC



HAND1



HAO1



Haptoglobin



HARS



HARS2



HBF



hCGÎ±



hCGÎ2



hCGÎ24



HCN4



HDAC1



HDAC10



HDAC2



HDAC3



HDAC4



HDAC6



HDAC9



HDHD1A



HDHD2



HDJ2



HDLBP



HE4



HEC1



HEF1



Helios



Hematopoiesis related Macrophage



Hematopoietic Lineage Cocktail



Hematopoietic Progenitor Cell



Hemoglobin



Hemoglobin F



Hemoglobin subunit Î±



Hepatitis B Virus



Hepatitis B Virus Core Antigen



Hepatitis B Virus E Antigen



Hepatitis B Virus Surface Antigen (Ad/Ay)



Hepatitis C Virus



Hepatitis C Virus Core Antigen



Hepatitis C Virus NS4



Hepsin



HER3



HER4



Hes1



Hexokinase



Hexokinase1



Hexokinase2



HFE1



HGF



HGFA Inhibitor 1



HHEX



HHV8 GPCR



HIBCH



HID1



HIF-1Î±



HIF-2Î±



HIF1AN



HINT1



HIP2



HIPK2



Hippocalcin



Histamine H3 Receptor



Histocytes



Histone H1



Histone H1.0



Histone H2A



Histone H2B



Histone H2B type 1B



Histone H3



Histone H3 Phospho (Ser10)



Histone H3 Phospho (Ser28)



Histone H3.3



Histone H4



HIV1 Core Antigen



HIV1 p17



HIV1 p24



HIV1 p55/p17



HIV1 tat



HL60



HLA Class I



HLA-2Kb/2Db



HLA-2kb/2Dd



HLA-A



HLA-A/B/C



HLA-A1/A11/A26



HLA-A1/A36



HLA-A10/A11



HLA-A10/A28/B75



HLA-A10/B62/B71



HLA-A11



HLA-A2



HLA-A2/A25/A32



HLA-A2/A28



HLA-A2/A3/A29



HLA-A2/A69



HLA-A2/B17



HLA-A2/B5



HLA-A2/B57



HLA-A23/A24



HLA-A24/A11/A2403



HLA-A25



HLA-A25/A26



HLA-A25/A26/A34



HLA-A25/A32



HLA-A26/A34/B71/B62



HLA-A29



HLA-A3



HLA-A30/A31



HLA-A33/B8



HLA-A34/B71/A26



HLA-A9



HLA-A9/A25/A32



HLA-A9/A32/B13



HLA-B



HLA-B12



HLA-B13/B62/B15



HLA-B14



HLA-B17



HLA-B17/B35/B44



HLA-B21/B70/B55



HLA-B27/B44/B47



HLA-B35/B57/B75/B77



HLA-B44/B75/B17



HLA-B48/B60



HLA-B5/B49/B56



HLA-B7



HLA-B8



HLA-B8/B14



HLA-BC



HLA-Bw4/A9/A32



HLA-Bw6



HLA-Bw6/B77



HLA-class I free chain



HLA-D



HLA-DM



HLA-DO



HLA-DP



HLA-DQ



HLA-DQ/DR



HLA-DQ1/DQ3



HLA-DQ1/DR7



HLA-DQ3



HLA-DQ6



HLA-DQ7



HLA-DQA1



HLA-DQB1



HLA-DQw1



HLA-DR



HLA-DR/DP



HLA-DR/DP/DQ



HLA-DR1



HLA-DR11



HLA-DR3/DR6



HLA-DR4



HLA-DR7



HLA-DR7/DRÎ2



HLA-DR8/DR12



HLA-DR9



HLA-DRA



HLA-DRÎ2



HLA-DRÎ23



HLA-E



HLA-G



HLCS



HLF



HLXB9



HMG14



HMG17



HMG4



HMGB1



HMGB2



HMOX1



HMOX2



HNF4Î±



hnRNPA1



hnRNPC1/C2



hnRNPD



hnRNPK



hnRNPL



hnRNPU



hnRNPUL1



Homing Receptor



HOXB4



HOXB5



HP1Î±



HPa1



HPa2



HPD



HPd1



HPd2



HPi1



HPi2



HPi3



HPi4



HPR1



HPRT1



HPV16 E1/E4



HPx1



HPx2



Hrk



Hsc70



HSD17B1



HSD3B1



HSF1



HSF2



HSF4



HSL



Hsp105



Hsp14



Hsp22



HSP25



Hsp27



Hsp40



Hsp47



Hsp60



Hsp70



Hsp70-2



Hsp90



Hsp90Î±



Hsp90Î2



HspA4



HspA6



HSPA9



HspB2



HspB7



HSV tag



HTLV I gp46



HTLV I p19



HtrA2/Omi



Human Papillomavirus 16 (E7)



Huntingtin



HUS1



Hydrogen Potassium ATPase I2



I-Ak (AÎ ± k)



I-Ak (AÎ2k)



Ia (B cells)



IBA1



IBP2



ICAD



IDO



IFABP



IFN-Î±



IFN-Î ± 1



IFN-Î ± 2Î2



IFN-Î2



IFN-Î3



IFN-Î3RÎ2



IFN-Î ©



IFNA1



IFNAR1



IFT88



Ig



Ig (polyspecific)



Ig light chain Î°



Ig light chain Î  



Ig light chain Î   1, Î   2, Î   3



IgA



IgA (Fab2)



IgA (H)



IgA, Î°



IgA, Î  



IgA1



IgA2



IgD



IgD (Î′ heavy chain)



IgDa



IgDb



IgE



IgE, Î°



IgEa



IgEb



IgG



IgG (Fab H/L)



IgG (Fab)



IgG (Fab2 Fc)



IgG (Fab2 H/L)



IgG (Fab2)



IgG (Fc)



IgG (H/L)



IgG (Î3 chain specific)



IgG Fd



IgG light chain



IgG, Î°



IgG/IgM



IgG/IgM/IgA



IgG/IgM/IgA (Fab2 H/L)



IgG/IgM/IgA (Fab2)



IgG/IgM/IgA (H/L)



IgG/IgY



IgG1



IgG1 (heavy chain)



IgG1, Î°



IgG1, Î  



IgG1/2a



IgG1/3



IgG1a



IgG1b



IgG2



IgG2, Î°



IgG2, Î  



IgG2/3



IgG2a



IgG2a, Î°



IgG2a, Î  



IgG2a/b



IgG2b



IgG2b, Î°



IgG2c



IgG2c, Î°



IgG3



IgG3, Î°



IgG3, Î  



IgG4



IgGDa



IgK



IGKC



IgL



IGLC2



IgM



IgM (Fab2)



IgM (Fc)



IgM (H/L)



IgM, Î°



IgM, Î  



IgMa



IgMb



IgY



Igâ    TMs



Ihh



Ikaros



IkBÎ±



IkBÎ2



IkBÎ¶



IKKÎ±



IKKÎ2



IKKÎ3 p(S376)



IKKÎμ



IL-10



IL-11RÎ±



IL-12



IL-12 (p35)



IL-12 (p70)



IL-12 RÎ21



IL-12 RÎ22



IL-12/IL-23 (p40)



IL-13



IL-15



IL-15/IL-15R



IL-15RÎ±



IL-16



IL-17D



IL-17A



IL-17A/F



IL-17B



IL-17C



IL-17E



IL-17F



IL-18



IL-18BP



IL-19



IL-1RA



IL-1RN



IL-1Î±



IL-1Î2



IL-2



IL-20R2



IL-20RÎ±



IL-20RÎ2



IL-21



IL-22



IL-22RÎ ± 2



IL-23 (p19)



IL-23R



IL-24



IL-25



IL-27



IL-27 (p28)



IL-27RÎ±



IL-28



IL-28RÎ±



IL-29



IL-3



IL-31



IL-32Î ± Î2Î3Î′



IL-32Î ± Î2Î′



IL-33



IL-34



IL-4



IL-4RÎ±



IL-5



IL-6



IL-7



IL-7RÎ±



IL-8



IL-9



ILF3



ILK



ILK1



ImmunofluorescenceN-Î3



IMP3



Importin9



Influenza A Virus M2 Protein



Influenza B Virus Nucleoprotein



ING1



ING2



ING3



ING4



Inhibin Î±



iNOS



INPP4A



INPP4B



Insulin



Insulin Degrading Enzyme (IDE)



Insulin Receptor R



Integrin Î ± 4/Î27



Integrin Î ± 9/Î21



Integrin Î ± V/Î25



Integrin Î ± V/Î26



Integrin Î21 Phospho (Tyr783)



Integrin Î21 Phospho (Tyr795)



Integrin Î25



Integrin Î26



Integrin Î27



Intercalated DNA



Intra Acrosomal Protein



Intra-Acrosomal Proteins



Invariant NK T



IP10



IQGA1



IRAK1



IRAK3



IRAK4



IRE1



IRF1



IRF3



IRF4



IRF5



IRF6



IRF7



IRF7 (pS477/pS479)



IRF8



IRF9



IRS1



IRS1 (pY896)



IRS2



IRS4



ISG15



ISG20



ISL1



Isthmin1



ITCH



Integrin Î ± 7



ITK



ITPR1



Jagged2



JAK2



JAK3



JAM2



JAML



Japanese encephalitis virus NS1 glycoprotein



JNK



JNK Phospho (Thr183/Tyr185)



JNK1/JNK2/JNK3



JNK2



Junctional Adhesion Molecule C



Junctophilin-1 (C-term)



Junctophilin-1 (Mid)



Junctophilin-2 (C-term)



Junctophilin-3 (C-term)



KAP1



KATNA1



KCNH1



KDEL



KDM4D



Ki-67



KIF22



KIF3A



KIF4A



KIFA3



Kindlin2



Kinetoplastid Membrane Protein 11 (KMP-1))



KIR-2.1



KIR-2D (pan CD158)



KLF4



KLF6



KLH



KLHL11



KLRA3



KLRC1



KLRG1



KMT4



KMT5A



KOR-SA3544



KS1/4



Ksp37



KSR1



Ku70



Ku70/80



Ku80



Kudoa Thyrsites



Kunitz Protease Inhibitor



Kv4.2



L/S-MAG



Labeling Check Reagent



Lactate Dehydrogenase



Lactate Dehydrogenase B



Lambda



Lamin A



Lamin A/C



Lamin B Receptor



Lamin B1



Lamin B2



Lamin C



Laminin



Laminin 5



Laminin Receptor



Laminin Î21



LAMP2a



LAMP2b



LAT



LAT (pY171)



LAT (pY226)



LBP



LC3



LC3B



LCAT



Lck



Lck (pY505)



LDH1



LDH1/B/C



LDL (MDA oxidized)



LDLR



LEF1



Leishmania LPG (repeat epitope)



Leishmania Major Surface Protease (GP-63)



LEKTI



Leukemia Inhibitory Factor



Leukotriene A4 hydrolase



Leukotriene B4 Receptor



LHX3



LI-Cadherin



LIF



DNA Ligase I



DNA Ligase III



LIM kinase 2



LIME1



LIMK1



LIMS1



Lin28



Lineage Cocktail



Lipin 1



LIS1



Liver Carboxylesterase 1



LKB1



LMO2



LOX



LOX1



LRP5/6



LRP6



LRPAP1



LSD1



LSP1



LSS



LTÎ±



Luciferase



LXRÎ±



Ly-108



Ly-49A



Ly-49A/D



Ly-49AB6



Ly-49C/F/I/H



Ly-49C/I



Ly-49D



Ly-49E/F



Ly-49F



Ly-49G



Ly-49G2



Ly-49G2B6



Ly-49H



Ly-49I



Ly-51



Ly-6A.2/Ly-6E.1



Ly-6A/E



Ly-6b



Ly-6B.2



Ly-6C



Ly-6D



Ly-6G



Ly-6G/C



Ly-6K



Ly-77



Lymphotoxin Î2



Lymphotoxin Î2 Receptor



Lyn



LYRIC



Lysophospholipase 1



Lysosomal acid lipase



Lysozome



Lysozyme



Lyve1



M-CSF



M13 Bacteriophage Coat Protein g8p



M13 Bacteriophage Protein



MAA



Mac-2BP



macroH2A.1



Macrophage



Macrophage Activator



Macrophage galactose lectin



Macrophage/Granulocyte



Macrophages/Monocytes



MAD2



MadCAM1



MADD



MADH7



MAFB



MAG



MAGE-A



MAGE1



MAIR2



MAIR4



MALT1



Mammaglobin A



MAP1LC3A



MAP2



MAP2B



MAP2K1IP1



MAP3K8



MAP4 Phospho (Ser768)



MAP4K1



MAP4K4



MAPK12



MAPK6



MAPKAP Kinase 2



MAPKAP Kinase 2 Phospho (Thr334)



MARCKS



MARCO



Marginal Zone B Cells



MARK2



MARK3



MART1



Mast Cell



Mast Cell Protease 11



mature macrophage marker



MBD1



MBD2



MBL



MCL1



MCM2



MCM3



MCM4



MCM5



MCM6



MCM7



MCP-1



MCP-4



MCP-8



MCSF



MD1



MD2



MDC



MECT1



MEF2A



MEIS1



MEK1



MEK1 (p298)



MEK1 (pS218)/MEK2 (pS222)



MEK1/2 (pS222)



MEK2



MEK3



MEK4



MEK5



MEK6



MEK7



MEKK1



MEKK2



MEKK3



MEKK4



Melanoma



MELK



MEMO1



Mena



Menin



MEOX2



Merlin



MERTK



Mesothelin



Metallothionein



MetRS



mGluR5



MGMT



MHC Class I



MHC Class I (H-2Db)



MHC Class I (H-2Dd)



MHC Class I (H-2Dk)



MHC Class I (H-2Dq/Lq)



MHC Class I (H-2Kb)



MHC Class I (H-2Kb/Db)



MHC Class I (H-2Kb/Dd)



MHC Class I (H-2Kd a3 domain)



MHC Class I (H-2Kd)



MHC Class I (H-2Kd/Dd)



MHC Class I (H-2Kd/Dd/q/u/v)



MHC Class I (H-2Kk)



MHC Class I (H-2Kq)



MHC Class I (H-2Ks)



MHC Class I (H-2Ld)



MHC Class I (H-2Ld/Db)



MHC Class Ib (H2-M3)



MHC Class II



MHC Class II (DQ)



MHC Class II (DR)



MHC Class II (I-A)



MHC Class II (I-A/E)



MHC Class II (I-Ab)



MHC Class II (I-Ab/Ad)



MHC Class II (I-Ab/As)



MHC Class II (I-Ad)



MHC Class II (I-Ak)



MHC Class II (I-Ak/Ad/Ab/Aq/Ar)



MHC Class II (I-Ak/As)



MHC Class II (I-Ap)



MHC Class II (I-Aq)



MHC Class II (I-E)



MHC Class II (I-EÎ°)



MHC Class II (RT1B)



MHC Class II (RT1Bu)



MHC Class II(RT1D)



MHC Class II Î2



MHC Qa1b



MICA



MICA/MICB



MICB



Microfold (M) Cells



Microtubule Associated Protein 2ab



Microtubule Associated Protein RP/EB 2



Midkine



Mineralocorticoid Receptor



MIP-1Î2



MIPEP



Mitochondria



Mitofilin



Mitofusin 1



Mitofusin 2



Mitotic Cells



MKK6



MLH1



MLK3



MLL1



MLLT11



MMP1



MMP10



MMP11



MMP12



MMP13



MMP14



MMP15



MMP17



MMP19



MMP2



MMP20



MMP21



MMP26



MMP3



MMP8



MMP9



Mnk1



mNOS



MnSOD



Moesin



Monoamine Oxidase B



Monocyte/Granulocyte



Mononuclear Phagocyte



Mouse Embryonic Fibroblast (mEF) Feeder Cells



Mouse Lineage



MPP1



MRCL3



MRE11



MRGPR-X2



MRI1



MRP14



MRP2



MRP3



MRP4



MRP5



MRP6



MRP8



MRP8/14



MSC (W8B2)



MSC (W3D5)



MSC (W5C5)



MSC (W7C6)



MSC/NPC



MSH2



MSH6



MSI2H



MSK1



MST1



MST1/MST2



MST3



MST4



MST4/MST3/STK25



mTOR



Muc-16



Muc-2



Muc-3



Muc-4



Muc-7



MULT-1



Munc13-4



Munc18



MUPP1



Mus81



Musashi1



Muscarinic Acetylcholine Receptor 2



muscle Actin



Muscleblind-like 1



MVP



MYBBP1A



MYBPC3



Myc tag



MyD88



Myelin Basic Protein



Myelin oligodendrocyte glycoprotein



Myelin PLP



Myeloid Antigen



Myeloid Cell Nuclear Differentiation Antigen



Myeloid Lineage



Myocilin



Myogenin



Myosin heavy chain



Myosin IIA



Myosin light chain 2



Myosin light chain 3



Myosin light chain kinase



Myosin Phosphatase



Myosin Phosphatase 1/2



MYST2



NADH2



Naf1



NAK



Nanog



NAPE-PLD



NAT1



Native Lipoteichoic Acid



Natriuretic Peptide Receptor A



Natural Killer Cell



Natural Killer Cell Activation Structures



NBS1



NC1.1



NCF4



Nck



NCOA1



NCOA2



NCX1



NDUFAF1



NDUFB4



NDUFS3



NEDD8



NEK2



NEK6



NEK7



NEK9



NEK9 Phospho (Thr210)



Nestin



NETO2



Neurabin1



Neuregulin1



Neuregulin3



Neuroblastoma



NeuroD1



NeuroD2



Neurofibromin



Neurofilament Heavy Protein



Neurofilament Medium Protein



Neurogenin 2



Neurokinin 1 Receptor



Neuron Specific Enolase



Neuronal Growth Factor Receptor



Neurotensin Receptor 1



NFÎ°B p50/p105



NFÎ°B p65 (pS536)



NFATc1



NFÎ°B p50



NFÎ°B p50/p105



NFÎ°B p52/p100



NFÎ°B p65



NFÎ°B p65 (pS529)



NG2



NGF



Nhedc2



NHERF1



Nicastrin



Ninein



Nitrotyrosine



NKG2A/C/E



NKG2AB6



NKp80



NKX3.1



NM23A



NMDA Receptor 2A



NMDA Receptor 2B



NMDE2



NMDZ1



NMNA2



nMyc



nNOS



NNTM



Nociceptin



Nod2



Nodal



Noggin



NONO



Nonspecific Cytotoxic Cells



Notch1



Notch2



Notch3



Notch4



NOX2



NOX4



NOXA2



NPC



NPM-ALK



NPM/B23 Phospho (Thr199)



NPM/B23 Phospho (Thr234/Thr237)



NPY5R



NQO1



NR2E1



NRC2C



Nrf2



NRG3



NSPA/B



NTAL



NTF97



Nucleolin



Nucleolin Phospho (Thr76/Thr84)



Nucleophosmin



NUDC



NUMA1



Nur77



O acetyl GD3



Oct2



Oct3/4



Oct3/4A



Oct4



ODAG



OGDH



OLIG1



OLIG2



Oligodendrocyte Marker



Oligodendrocyte Marker O1



Oligodendrocyte Marker O4



Oncostatin M Receptor



Orai1



OSCAR



OSR1



Osteonectin



Osteopontin



Osteoprotegerin



Otx2



OVA (SIINFEKL) H-2Kb



Oval Cell Marker



Ovalbumin



Ovarian Carcinoma-associated Antigen



OX-62



p110Î′



p120 Catenin



p120 Catenin (pS268)



p120 Catenin (pS288)



p120 Catenin (pS879)



p120 Catenin (pT310)



p120 Catenin (pT916)



p120 Catenin (pY228)



p13



p130



p130 Cas



p130 Cas (pY249)



p14ARF



p150, 95



p19ARF



p21



p22phox



p23



p27Kip1



P2RX4



P2RY8



P2X3



P2X7



P2Y6



p34Cdc-2



p38



p38 MAPK (pT180/pY182)



p400



p53



p53 Acetylated (Lys305)



p53 Acetylated (Lys382)



p53 Phospho (Ser15)



p53 Phospho (Ser37)



p53 Phospho (Ser392)



p53BP1 (Ser1778)



p57Kip2



p60 CAF1



p62



p63



p63 (TA)



p70 S6 Kinase Î2



p90 Rsk



p90 Rsk Phospho (Thr368/Ser372)



p95 NBS1



p97



PA28Î3



PABP1



PABP2



PABPN1



PAC1



PAD2



PAG1



PAK1



PAK2



PAK3



pan Actin



pan Macrophage



Panendothelial Cell Antigen



PAR1



Parainfluenza Virus type 1



Parainfluenza Virus type 2



Parainfluenza Virus type 3



PARC



PARD3



PARK7/DJ1



PARP, Cleaved Form



PARP16



PARP4



PARVA



Pax2



Pax5



Pax6



Pax7



Pax8



Pax9



Paxillin



Paxillin Phospho (Tyr118)



Paxillin Phospho (Tyr31)



PBEF



PBK



PBP



PBR



PBX3



PCB



PCNA



PCYT1A



PD-1H



PD-ECGF



PDC-TREM



PDCD4



PDCD6



PDE3B



PDECGF



PDGF-AA



PDI



PDK1



PDK2



PDPK1



PDPK1 (pS241)



PDX1



PDZK1



PE



PECR



PEI-Transferrinfection



Pellino 1



Pentraxin 3



PEPD



Perforin



Peroxiredoxin 1



Peroxiredoxin 2



Peroxiredoxin 6



PEX5



PF4



PGC1Î±



PGIS



PGP9.5



PGRP-Ia



PGRP-S



PHD1



PHD2



Phosphatidylserine



Phospho SHIP



Phospholipase A2 activator protein (PLAP)



Phospholipase C Î23



Phospholipase C Î31



Phospholipase D1



Phosphoserine/threonine/tyrosine



Phosphotyrosine



PI 3 Kinase catalytic subunit Î±



PI 3 Kinase catalytic subunit Î3



PI 3 Kinase p110 Î2



PI 3 Kinase p110 Î′



PI 3 Kinase p150



PI 3 Kinase p85 Î±



PI 4 kinase Î2



PIAS1



PIAS3



PICK1



PIM1



PIM2



Pin1



PINK1



PIP5K2Î±



PIP5KIÎ3



PIR-A/B



Pirh2



PIST



PiTX3



PIWIL2



PKA RIIÎ± (pS99)



PKA RIIÎ2 (pS114)



PKA2Î2



PKAR2



PKAÎ3



PKC



PKCq



PKCÎ±



PKCÎ± (pT497)



PKCÎ± (pT638)



PKCÎ2



PKCÎ22



PKCÎ3



PKCÎ′



PKCÎμ



PKCÎ¶



PKCÎ,



PKCÏ . . .



PKN



PKN2



PKR



PKX1



PLA2G1B



Placental alkaline phosphatase



Placental Protein 14



Plakophilin 3



Plastin L



Platelet



PLAU



PLCÎ31



PLCÎ31 (pY783)



PLCÎ32



PLCÎ32 (pY759)



Plectin



Pleiotrophin



PlexinA1



PlexinB2



PLGF



PLK1



PLK1 Phospho (Thr210)



PLK4



PLSCR1



PLVAP



PLZF



PMCA(1-4)



PMCA4



PMEL17/SILV



PMN



PMP70



PMS2



PNAd



PNPH



Podocalyxin



Podoplanin



POKEMON



Polyhistidine Tag



PON1



PON3



PP2AÎ±



PP2AÎ ± Î2



PPM1A



PPP1A



PPP5C



PPP6C



PR3



PRA1



PRC1



Pre-BCR



Pre-T Cell Receptor Î ± Chain



Prealbumin



Presenilin1



Presenilin2



Prion protein PrP



PRKRA



PRLR



PRMT1



PRMT5



pro Relaxin 1/2



pro Relaxin 2



Profilin1



Progesterone Receptor



Prohibitin



Prokineticin 1



Prokineticin 2



Prolactin



ProMBP1



Prostaglandin D2 Receptor



Prostaglandin dehydrogenase 1



Prostaglandin E Receptor EP3



Prostate Cell Surface Antigen



Prostate Specific Antigen



Prostatic Acid Phosphatase



Proteasome 20S C2



Proteasome 20S Î ± 2



Proteasome 20S Î ± 3



Proteasome 20S Î ± 5



Proteasome 20S Î ± 6



Proteasome 20S Î ± 7



Proteasome 20SÎ ± 1/2/3/5/6/7



Protein A



Protein G



Protein Kinase D2



Protein Phosphatase 1Î2



Protein phosphotase inhibitor 1



Protein S



Proteinase Activated Receptor 4



Prothrombin



PSA-NCAM



PSD95



Pseudomonas Aeruginosa



PSMA



PSMD14



Psoriasin



PTAFR



PTBP1



PTEN



PTGER2



PTGER4



PTHLH



PTK7



PTP1B



PTP4A2



PTPS



PTPÎ1⁄4



PTRH2



PU.1



PU60



PUMA



PUMAÎ3



Pumilio1



Pumilio2



PXR



PYCARD



Pygopus2



Pyk2



Pyk2 (pY402)



Pyruvate Dehydrogenase E1Î±



Pyruvate Dehydrogenase E2



Pyruvate Dehydrogenase E2/E3bp



q2



Qa1(b)



Qa2



RAB11A



RAB25



RAB27A



RAB4



RAB5a



RAB9



Rac1



Rac1/Cdc42



RAD17



RAD17 Phospho (Ser645)



RAD23A



RAD51



RAD54



RAD9A



Radixin



RAE-1Î3



RAE-1Î′



RAF1



RAGE



RAIDD



Rainbow Trout Ig



RalBP1



RanBP9



RanGAP1



RAP1A/RAP1B



RAP1GAP



Raptor



RARÎ±



RAS



RASGAP



RASGRF1



RASSF1A



Rb



Rb (a.a. 332-344)



Rb (pS780)



Rb (pS807/pS811)



RbAp46



RbAp48



RBC



RBC (Polyclonal Rabbit)



RBM35A



RBP4



RBX1



RCC1



RcRL6



Red Blood Cell



Relaxin 1



Relaxin 1/2



Relaxin 2



RelB



RELMÎ2



RELT



Renin



RENT1



Reptin



Repulsive Guidance Molecule C



Resistin



REST



Ret



Reticular Fibroblasts and Reticular Fibres



Reticulon1A



Reticulum Cells



Retinoblastoma 1



RFLAT1



RFP



RGS6



RGS7



RGS9



RHEB



Rho



RhoA



RHOC



RhoGAP



RhoGDI



RIAM



RICTOR



RIG1



RIP1



RIP2



Rituximab



RLA DQ



RLA DR



RNA polymerase II



RNA polymerase II CTD repeat YSPTSPS



RNASE-L



RNASE1



RNF144B



RNF168



RNF36



RNPEP



ROCK1



ROR1



ROR2



RORÎ±



RORÎ3



ROS



RPA32/RPA2



RPA70



RPS6



RSF1



RSK1 p90



RSK2



RSK3



RSK4



RT1A



RT1Aa



RT1Aa, b



RT1Aa, b, l



RT1Ac



RT1Au



RT1B



RT6.1



RT6.2



Ryanodine Receptor



RYK



RyR



S-Tag



S100A1



S100A10



S100A13



S100A4



S100A6



S100A9



S100Î±



S100Î ± 2



s100Î2



S6 (pS235/pS236)



S6 (pS240)



S6 (pS244)



S6K



SAA4



Sall4



Salmonella Paratyphi A



Salmonella Typhimurium



Salmonid Ig (H and L chain)



Salmonid Ig (H chain)



SAM68



SAMD2



SAP



SARA



SATB1



SATB2



SC5A5



SC6A4



SCAI



SCD1



Scramblase1



SCY1-like 3



SDF1



SDF1Î±



SDHA



SDHB



Secretory component



Securin



SELP



Sema4A



Sema7A



SENP1



SEPP1



SERCA2



SerpinB1



SerpinB2



SerpinB6



Sestrin1



SFRP2



SGK1



SHC1



Shigella Boydii



SHIP1



SHP1



SHP2



SHP2 (pY542)



SIAH2



SIGIRR



Siglec-10



Siglec-8



Siglec-9



Siglec-F



Siglec-H



SIK2



SIRT1



SIRT2



SIRT3



SIRT5



SIT1



SIX2



SKP1A



SLA-DR



Slan



SLC1A3



SLC1A7



SLC22A1



SLC22A5



SLC26A6



SLC26A7



SLC30A4



SLC39A11



SLC4A3



SLC6A19



SLC6A6



SLC7A10



SLC7A14



SLC7A3



SLC7A8



SLC8A2



SLC9A6



SLP76



SLP76 (pY128)



SM22Î±



SMAC



SMAC3



SMAD1



SMAD1 (pS463/465)



SMAD1/5



SMAD1/9



SMAD2



SMAD2/3 (pS465/467) DELETE



SMAD3



SMAD4



SMAD5



SMAD6



SMC1



SMC1L1



SMN



Smoothelin



SMURF2



SNAP25



SNX1



SOAT1



SOCS1



SOCS2



SOCS3



SOCS6



SOD2



Sodium Potassium ATPase



Sonic Hedgehog



Sortilin



SOSC3



SOX1



SOX10



SOX17



SOX18



SOX2



SOX2 (COOH terminus)



SOX2 (NH2 terminus)



SOX9



SP-D



Sp1



Sp3



Spectrin Î ± 1



SPHK1



Spt16



Src (pY418)



SREBP1



ssDNA



SSEA3



SSEA4



SSEA5



SSH3BP1



SSR2



SSR5



SSRP1



SSX2IP



Stat1



Stat1 (N-Terminus)



Stat1 (pS727)



Stat1 (pY701)



Stat1Î±



Stat2



Stat3



Stat3 (pS727)



Stat3 (pY705)



Stat4



Stat4 (pY693)



Stat5



Stat5 (pY694)



Stat5a



Stat5b



Stat6



Stat6 (pY641)



Stathmin/Op18 Phospho (Ser16)



Stathmin1



Stefin B



Stem Cell Factor



STIM1



STK3



STK33



STK39



STOM



STRO1



STUB1



SULT1A1



SULT1A3/SULT1A4



SULT1C2



SULT2A1



SUMO1



SUMO2



SUMO3



SUN1



Suppressor of Fused



SUPT16H



Survivin



Survivin Phospho (Thr34)



SV40 Large T and Small t Antigens



SWC1a



SWC6



SYBL1



Syk



Syk (pY348)



Synapsin I



Synapsin II



Synaptojanin2



Synaptophysin



Syndecan4



SynGAP



Synip



Syntaxin



Syntaxin6



Syntrophin



SYWC



T cells (pan reactive)



T Lymphocytes



T- and B-Cell Activation Antigen



T7 tag



TAB1



TACE



TACI



TAF172



TAF250



TAG72



Talin1



Talin2



Tamm Horsfall (Uromucoid)



TANK1



TAP1



TAP2



TARDBP



TARP



Tartrate-resistant acid phosphatase



TAS1R1



Tau



TBA1B



Tbet



TBK1 (pS172)



TBX1



TC10



TCF3



TCF7L1



TCF7L2



TCL1



TCP1Î±



TCP1Î2



TCR



TCR DO11.10



TCR HY



TCR VÎ ± 11



TCR VÎ ± 11.1/11.2b, d



TCR VÎ ± 2



TCR VÎ ± 24



TCR VÎ ± 24-JÎ ± 18



TCR VÎ ± 3.2



TCR VÎ ± 3.2b, c



TCR VÎ ± 7.2



TCR VÎ ± 8



TCR VÎ ± 8.3



TCR VÎ21



TCR VÎ210a



TCR VÎ210b



TCR VÎ211



TCR VÎ212



TCR VÎ212b



TCR VÎ213



TCR VÎ213.1



TCR VÎ213.2



TCR VÎ213.6



TCR VÎ214



TCR VÎ216



TCR VÎ217



TCR VÎ217Î±



TCR VÎ218



TCR VÎ22



TCR VÎ220



TCR VÎ221.3



TCR VÎ222



TCR VÎ223



TCR VÎ23



TCR VÎ24



TCR VÎ25



TCR VÎ25.1



TCR VÎ25.1/5.2



TCR VÎ25.2



TCR VÎ25.3



TCR VÎ26



TCR VÎ27



TCR VÎ27.1



TCR VÎ27.2



TCR VÎ28



TCR VÎ28.1/8.2



TCR VÎ28.2



TCR VÎ28.2/8.3



TCR VÎ28.2/8.4



TCR VÎ28.3



TCR VÎ28.5



TCR VÎ29



TCR VÎ31.1



TCR VÎ31.1/Î31.2



TCR VÎ32



TCR VÎ33



TCR VÎ39



TCR VÎ′1



TCR VÎ′2



TCR VÎ′4



TCR VÎ′6.3/2



TCR Î±



TCR Î ± Î2



TCR Î2



TCR Î3Î′



TCR Î¶



TCTP



TdT



Tec



TEF1



TEM8



Tenascin C



TER119



TERF2



Terminal-Deoxynucleotidyl Transferase



TERT



Tetranectin



TFF3



TFIIB



TGF-Î2



TGF-Î21



TGF-Î23



TGF-Î2R1



TGF-Î2R2



TGN38



TGN46



THAP11



THEMIS



Thioredoxin



Thioredoxin Reductase 1



ThPOK



Thrombin Receptor



Thrombocyte



Thrombospondin



Thymidine Kinase 1



Thyroglobulin



TIA-1



TIAM2



Tie1



Tie2 (pY1102)



Tie2 (pY992)



TIF1Î2 Phospho (Ser473)



TIGIT



Tim1



Tim2



Tim3



Tim3 Fc Fusion Protein



Tim4



Tim50



Timeless



TIMP1



TIMP2



TIP49A



TIRAP



TIS11b



TL1A



TLK1



TLR11



TLR12



CD285



TLR7



TLR8



TMEFF2



TMPS2



TMSA



TMTSP



TNAP



TNAP3



TNF-Î±



TNF-Î2



TNFR Related Protein



TNPO3



Tollip



TOMM20



TOMM22



TOP1



TOP2A



TOP2B



TORC2



Torsin A



TOX



TPH1



TPPP



TPTE



TR11B



TRA-1-60



TRA-1-60R



TRA-1-81



TRA-2-49



TRA-2-54



TRADD



TRAF2



TRAF4



TRAF5



TRAF6



TRAM2



Transferrin



Transglutaminase



Transglutaminase2



Transketolase



TRAP1



TRAPPC2



TRAPÎ±



Trem-like 2



Trem-like 4



TRIB2



TRIB3



TRIM



TRIM25



TRIM29



TRK



TrkA



TrkC



Trop2



Tropomyosin 1



TROY



TRPC6



TRPM2



TRPM8



TRX1



Trypanosoma brucei Major Lysosomal Protein



Trypanosoma brucei procyclin (EP)



Trypanosoma congolense procyclin



Trypanosoma cruzi LPG



TSC2 Phospho (Ser664)



TSC2 Phospho (Thr1462)



TSG101



TSHR



TSLP



TSLP Receptor



TSPO



TTF1



Tubb3



Tuberin



Tubulin Î±



Tubulin Î ± 1B



Tubulin Î ± 4a



Tubulin Î ± 3E



Tubulin Î ± 8



Tubulin Î2



Tubulin Î2 class III



Tubulin Î24



Tubulin Î3



tumor antigens of epithelial origin



Twist2



TXNIP



TYK2



TYMS



Tyro3



Tyrosinase



Tyrosine Hydroxylase



UACA



UBA52



UBC9



UBE2



UBE2L3



UBE2L6



UBE2M



UBE2N



UBF



UBF1



Ubiquitin



UBK63



UCH37



UCK



UCP2



UCP3



UFM1



ULBP1



ULBP2



ULBP4



ULK3



UNC5A



UNC5B



UNG



uPA



UQCRC1



UQCRC2



Urm1



URP2



USF1



USP11



USP13



USP22



USP28



USP7



UTF1



V5 tag



VAMP5/8



VAP1



VASA



VASP



VAV1



VAV2



VAV3



VDAC1



VEGF



VEGF-120



VEGF-A



VEGF-R1



VELIS-3



VGLU1



Villin



Vimentin



Vinculin



Viperin



VIPR1



Vitamin D Binding protein



Vitamin D Receptor



Vitronectin



VMAT2



vMyb/cMyb



von Willebrands factor



VRK1



VSV-G tag



WAPL



WASP



WC14



WC15



WCD44



WIP (pS488)



WNT1



WNT16



WNT2



WNT5B



WNT6



WSTF



WWOX



Xanthine Oxidase



XBP1



XBP1 (COOH terminus)



XBPs



XCL1



XIAP



XPC



XPNPEP3



XRCC2



XTP4



YAP1



YB1



YES1



YY1



ZAP-70



ZAP-70 (pY292)



ZAP-70 (pY319)



ZAP-70 (pY319)/Syk (pY352)



ZBP-1



ZIPK



ZO-1 (Mid)



ZONAB (Mid)



Zyxin



IL-33R



Globo H



CCL8



Siglec-G



CD307e



CLEC6



Snail1



SMAD1 (pS463/pS465)/SMAD8 (pS465/pS467)



SMAD2 (pS465/pS467)/SMAD3 (pS423/pS425)



GSK-3Î2 (pY216)



NKX6.1



FAK (pY397)



Btk (pY223)/Itk (pY180)



ERK3



CD276Î2



MCP-3



FcÂμR



CD238



beta2 Microglobulin [b, c]



Nucleostemin



GPR-49 (Central LRR)



GPR-49 (N-Terminal)



Phospholipase C Î24



coilin



HNF1Î2



Trinitrophenal



Annexin VII



CD301a



CD301b



mTOR (pS2448)



PI16



MSC (W5C5)



LAMP5



GPR-19



FPRL2



CXCL5



PAR2



PDGF-RÎ±



ULBP6



ULBP2/5/6



IL-17B Receptor



ULBP3



Arginase 1



Alkaline Phosphatase



ULBP3



TrkB



Osteocalcin



IL-22RÎ ± 1



APJ



IFN-Î±/Î2 Receptor Subunit 2



FGFR3



SR-A1



Rae-1 (pan)



CXCL12



TREM2



Brachyury



CLEC5A



Integrin Î ± 7



Mer



XCR1



AML2



von Willebrands factor A2



MMP7



GLP-1R



FR1



IL-1RAcP



Claudin-6



Leptin Receptor



Caherin 6



IL-1R type II



Nectin4



Delta like protein 3



ChemR23



GPR-39



CD158b2



IL-10RÎ±



LRIG1



Neuropilin2



IL-10RÎ2



IL-18RÎ2



GPR-44



Eph Receptor B2



Glypican3



IFN-Î3R2



IL-17C Receptor



BMPR1B



IL-31RA



OCIL



Frizzled-7



IL-26



GPR-15



PlexinD1



CD158



FPR1



HBEGF



Vitamin D3



PlexinB1



Somatostatin Receptor 2



OV-6



CXCL16



Siglec-E



EDG5



Ninjurin-1



Integrin Î ± 9



MHC Class II (I-Ed/j/k/p/r/u/v)



ThB



MAP-2 (2a & 2b)



IgM Î1⁄4-chain



MHC Class I (H-2b/p)



MHC Class I (H-2s/p/q/d/u/r)



MHC Class I (H-2s/f)



CDw60



Bad Phospho (Ser112)



Caspase 3 Cleaved (Asp175)



Chk1 Phospho (Ser345)



Chk2 Phospho (Thr68)



Cyclin D1 Phospho (Thr286)



cFos Phospho (Ser32)



FosB



GSK-3Î2 (pSer9)



Histone H3 Acetylated (Lys9)



HS1 Phospho (Tyr397)



Hsp27 Phospho (Ser82)



ID3



CD221Î2



Phospho-IRAK4 (Thr345/Ser346)



Phospho-cJun (Ser73)



S6 (pS240/pS244)



Syk (pY525/pY526)



C23



Hemoglobin Î2



CD221Î±



p27



cJun Phospho (Ser63)



PPARÎ3



ENPP1



PILRÎ±



PILRÎ2



Twist1



Cadherin M



CD302



CD66d



CLEC14A



CD242



Syndecan2



IL-32Î±



CDO



Cryptic



Endothelin B Receptor



FR3



IGSF3



CD85f



Matriptase



MCEMP1



mGluR4



Stabilin1



Stabilin2



Cadherin 13



GPR-109A



TSPAN8



Reg1A



Cadherin 12



ECE1



FABP5



IGSF4C



Trem-like 1



Activin A Receptor Type IIA



ALK7



BCAM



BLAME



CEACAM4



Claudin-3



CLP24



CRHR1



DC-STAMP



Eph Receptor B3



FATP4



FcRL1



FcRL2



FcRL3



FSH-R



Gi24



Histamine H1 Receptor



NeuSGc



Lin28A



IL-33RÎ±



ATM (pSer1981)



Integrin Î ± 8



Integrin Î27



Integrin Î28



CD158k



KOR



CD85i



LRIG3



LRP4



MMP16



MS4A4A



NAALADase-like 2



Neuropeptide Y receptor t ype 1



Oncostatin M Receptor Î2



MS4A3



PEAR1



PEDF Receptor



PlexinA4



Protocadherin1



ROBO2



ROBO4



EDG8



Scavenger receptor A5



Semaphorin 4A



Semaphorin 4B



Semaphorin 6A



Siglec-16



Somatostatin Receptor 3



STING



GPBAR1



TM4SF4



TMEM87A



TSPAN2



VEGF-R1, 2, 3



ADAM15



Calreticulin2



Complement Factor H-related 4



CXCL6



CD158a/h/b2/f/g



Ea52-68 peptide bound to I-Ab



HLA-Bw4



ATF1 Phospho (Ser63)



Epiregulin



FATP1



Fibromodulin



Furin



Galanin



IL-11



CD306



MFG-E8



MINA



Oct4A



OLIG1, 2, 3



Oncostatin M



Semaphorin 3E



Slug



SOX3



STYK1



LTBP1



TIMP3



VAP-B



WNT9a



5HT2C



AATK



ACLP



ADAMTS15



alpha 1B Adrenoreceptor



APLP1



Fluorescein/Oregon Green



RXR-Î2



L3MBTL3



CCL1



PRDM4



ACTH



PDZ binding kinase



HuC/HuD neuronal protein



TDRD3



EP300



Carbonic Anhydrase VI



Cholecystokinin A Receptor



CCL23



CD1e



Chondrolectin



Chordin-Like 2



Claudin-10b



Claudin-11



Claudin-12



Claudin-17



CLEC2A



Coagulation Factor VII



CXCL1/2/3



DDR2



DPCR1



Dipeptidyl peptidase 6



Epithelial membrane protein 3



Endoglycan



Calgranulin C



FATP2



FATP5



FcRLB



GLP-2R



GLUT3



Glypican6



GPR-22



GPR-37



GPR-37L1



INSRR



LINGO1



LINGO2



mGluR2



mGluR7



MMP25



Neuromedin B Receptor



NRAGE



Osteoactivin



Porimin



Prokineticin Receptor 1



Prominin2



Semaphorin 3A



SLAP-130



Somatostatin Receptor 5



SCARF1



STAMP2



TAFA3



TAFA4



TM4SF18



Tuberous Sclerosis 1



TCF8



CMG2



IL-17D Receptor



Macrophage Stimulating Protein Receptor



Siglec-11



Syndecan3



TGF-Î2R3



CD85e



SOX7



Activin A Receptor Type IA



Carbohydrate Sulfotransferase 15



CD300b



CELSR3



Coagulation Factor II



DC-SCRIPT



DSCAM-L1



FLRT1



Frizzled-6



Glypican1



IGSF4B



IL-1R9



BAZ2B



BRD4



Kell



Kremen2



LAX1



CD85c



MIF



Neprilysin2



OBCAM



PlexinC1



RGM-B



Wilmsâ    TM Tumor protein 1



Xg



DCBLD2



ASAM



Desmocollin1



Frizzled-3



MMP24



TOR



WNT3a



Glypican5



Jagged1/Jagged2



Pax3



CELSR2



Cyclin D1/D2



PlexinA2



TAFA5



FR4



CD315



NKG2I



RAMP2



TNFRH3



Biotin



GPVI



MS4A4B



PIR-B



Semaphorin 4F



IL-1F6



CD39L3



Contactin 3



CLEC4B



MC3R



PGRP-L



PLET1



ADAM9



AMIGO3



CD99-L2



Eph Receptor A5



Ephrin B2



CD316



Kremen1



Eph Receptor B1



PlexinB3



DMBT1



FcRn



LIMPII



MUCDHL



Patched1



SLC39A4



IGSF4A



PRAT4B



HHV8-ORF74



4E-BP1 Phospho (Thr36/45)



4E-BP1 Phospho (Thr69)



DCAR1



Von Hippel-Lindau



Isotype Control



Granzyme M



REA Isotype Control



CD300LG



MR1



CD327



B7-H6



CLEC4G



BATF3



IL-38



Monocarboxylic Acid Transporter 1



MC5R



TCF7



TM4SF1



GPR-49 (CRL Region)



CD156a



ADAM33



ADAMTS13



CCL16



CXCL17



Deltex1



FBXO15



GPR34



GPRC5A



Proinsulin



JAK1



MEP1A



Hypocretin receptor 2



p70S6K



RAE-1Îμ



STRA6



FcÎ3RIIA



Insulin R/IGF-I R Heterotetramer



SPARCL1



Spi-B



TRAM



Carboxypeptidase E



Islet Cell Autoantigen 1



Patched2



ST8SIA2



AML1 (pS249)



AMPKÎ21 (pS182)



BRF1/2



Histone H3 Phospho (Thr11)



MEK1 (pT286)



MMP16



MNK Phospho (T197/T202)



NUMB



Hsp27 Phospho (Ser78)



PKCÎ, (pT538)



SIRT1 (pS47)



ZAP-70 (pY493)



ZAP-70 (pY315/pY319)



sRAGE



mCherry



PI 3 Kinase regulatroy subunit Î±



TIMP4



SRC



ZAP-70 (pT493)



TSC2 Phospho (S939)



RagC



SHIP2



MKK4 (pS257)



CD79a (pY182)



TRAF1



EVI1



SRC3



SOX11



IL-17F homodimer



CCRL1



FOXP2



IFNAR2



REA Control



CD228



Muc-13



P2X7R



Btk (pY223/Itk (pY180)



CD248



GILT



Recoverin



Cardiac Troponin I



PTF1Î±



NKX2.2



HLA-B7/B27



Myosin light chain 2a



Myosin light chain 2v



Epithelial Antigen



CD79Î ± cy



CD92










In one embodiment, a plurality of hydrogel particles is used to determine the dynamic range and/or sensitivity of detection of a particular cell surface marker or combination thereof on a population of target cells. For example, the population of hydrogel particles can be tuned to have the SSC and/or FSC profile of the target cell, and subpopulations of the hydrogel particle are derivatized with a specific number of copies of a cell surface marker, e.g., a cell surface receptor, or a domain thereof, for example, an epitope binding region thereof. For example, individual subpopulations of hydrogel particles can each be derivatized to have a unique number of copies, e.g., one subpopulation will contain 100 copies of a cell surface marker, a second subpopulation will contain 1,000 copies of the same cell surface marker, a third subpopulation will contain 10,000 copies of the same cell surface marker, etc. The populations of hydrogel particles are fluorescently stained for the respective cell surface marker and fluorescence is detected for hydrogel particles in each subpopulation. In this regard, the subpopulations of hydrogel particles can be used to generate a standard curve of fluorescence emission for target cells with the respective cell marker. The cell surface marker can be any of the cell surface markers provided thereof, or binding regions thereof, or a cell surface marker known to one of ordinary skill in the art.
Hydrogel particles of the disclosure behave similarly to target cells in procedures such as staining and analysis by flow cytometry or FACS. For example, in one embodiment, a hydrogel particle has one or more optical properties substantially similar to one of the cell types set forth in Table 1, Table 2 or Table 3.
In some embodiments, a target cell is an immune cell. Non-limiting examples of immune cells include B lymphocytes, also called B cells, T lymphocytes, also called T cells, natural killer (NK) cells, lymphokine-activated killer (LAK) cells, monocytes, macrophages, neutrophils, granulocytes, mast cells, platelets, Langerhans cells, stem cells, dendritic cells, peripheral blood mononuclear cells, tumor infiltrating (TIL) cells, gene modified immune cells including hybridomas, drug modified immune cells, and derivatives, precursors or progenitors of any of the cell types listed herein.
In some embodiments, a target cell encompasses all cells of a particular class of cell with shared properties. For example, a target cell can be a lymphocyte, including NK cells, T cells, and B cells. A target cell can be an activated lymphocyte.
In some embodiments, a target cell is a primary cell, cultured cell, established cell, normal cell, transformed cell, infected cell, stably transfected cell, transiently transfected cell, proliferating cell, or terminally differentiated cells.
In one embodiment, a target cell is a primary neuronal cell. A variety of neurons can be target cells. As non-limiting examples, a target cell can be a primary neuron; established neuron; transformed neuron; stably transfected neuron; or motor or sensory neuron.
In other embodiments, a target cell is selected from the group consisting of: primary lymphocytes, monocytes, and granulocytes.
A target cell can be virtually any type of cell, including prokaryotic and eukaryotic cells.
Suitable prokaryotic target cells include, but are not limited to, bacteria such as E. coli, various Bacillus species, and the extremophile bacteria such as thermophiles.
Suitable eukaryotic target cells include, but are not limited to, fungi such as yeast and filamentous fungi, including species of Saccharomyces, Aspergillus, Trichoderma, and Neurospora; plant cells including those of corn, sorghum, tobacco, canola, soybean, cotton, tomato, potato, alfalfa, sunflower, etc.; and animal cells, including fish, birds and mammals. Suitable fish cells include, but are not limited to, those from species of salmon, trout, tilapia, tuna, carp, flounder, halibut, swordfish, cod and zebrafish. Suitable bird cells include, but are not limited to, those of chickens, ducks, quail, pheasants and turkeys, and other jungle foul or game birds. Suitable mammalian cells include, but are not limited to, cells from horses, cows, buffalo, deer, sheep, rabbits, rodents such as mice, rats, hamsters and guinea pigs, goats, pigs, primates, marine mammals including dolphins and whales, as well as cell lines, such as human cell lines of any tissue or stem cell type, and stem cells, including pluripotent and non-pluripotent, and non-human zygotes.
Suitable cells also include those cell types implicated in a wide variety of disease conditions, even while in a non-diseased state. Accordingly, suitable eukaryotic cell types include, but are not limited to, tumor cells of all types (e.g., melanoma, myeloid leukemia, carcinomas of the lung, breast, ovaries, colon, kidney, prostate, pancreas and testes), cardiomyocytes, dendritic cells, endothelial cells, epithelial cells, lymphocytes (T-cell and B cell), mast cells, eosinophils, vascular intimal cells, macrophages, natural killer cells, erythrocytes, hepatocytes, leukocytes including mononuclear leukocytes, stem cells such as hematopoietic, neural, skin, lung, kidney, liver and myocyte stem cells (for use in screening for differentiation and de-differentiation factors), osteoclasts, chondrocytes and other connective tissue cells, keratinocytes, melanocytes, liver cells, kidney cells, and adipocytes. In certain embodiments, the cells are primary disease state cells, such as primary tumor cells. Suitable cells also include known research cells, including, but not limited to, Jurkat T cells, NIH3T3 cells, CHO, COS, etc. See the ATCC cell line catalog, hereby expressly incorporated by reference.
In some embodiments, a target cell is a tumor microvesicle or tumor macrovesicle. Tumor microvesicles, also known as tumor-secreted microvesicles or tumor-secreted exosomes, can be found in circulating blood and may have immune-suppressive activities. Tumor microvesicles typically range in size from 30-200 nm in diameter. Larger tumor micro vesicles may be referred to as tumor macro vesicles, and can range in size from 3-10 μm in diameter.
The hydrogel particles described herein can be employed in any flow cytometer known to those of ordinary skill in the art. For example, one or more of the flow cytometers provided in Table 9 below are amenable for use with the hydrogels and assays described herein.







TABLE 9







Instruments for use with embodiments described herein








Instrument
Manufacturer





MACSQuant ® Analyzer 10
Miltenyi


MACSQuant ® VYB
Miltenyi


BD FACSCalibur TM
BD Biosciences


BD FACSCanto TM High Throughput Sampler
BD Biosciences


BD FACSCanto II
BD Biosciences


BD FACSCanto TM
BD Biosciences


BD FACSCount TM
BD Biosciences


BD Accuri TM C6
BD Biosciences


BD LSRFortessa TM X-20
BD Biosciences


BD FACSCanto TM II
BD Biosciences


BD LSR II
BD Biosciences


BD LSRFortessa TM
BD Biosciences


BD FACSVerse TM
BD Biosciences


BD FACSAria TM Fusion
BD Biosciences


BD FACSAria TM
BD Biosciences


BD FACSAria TM III
BD Biosciences


BD FACSJazz TM
BD Biosciences


BD Influx TM
BD Biosciences


Fortessa X50.
BD Biosciences


FlowSight Flow Cytometer
Millipore


Guava easyCyte 6-2L Benchtop Flow Cytometer
Millipore


guava easyCyte 5HT Benchtop Flow Cytometer
Millipore


guava easyCyte 8 Benchtop Flow Cytometer
Millipore


guava easyCyte 5 Benchtop Flow Cytometer
Millipore


guava easyCyte 8HT Benchtop Flow Cytometer
Millipore


guava easyCyte 6HT-2L Benchtop Flow
Millipore


Cytometer


ImageStreamX Mark II Imaging Flow Cytometer
Millipore


Muse Cell Analyzer
Millipore


guava easyCyte 12HT Benchtop Flow Cytometer
Millipore


guava easyCyte 12 Benchtop Flow Cytometer
Millipore


S3e TM Cell Sorter
Bio-Rad


S3 TM Cell Sorter
Bio-Rad


Avalon Cell Sorter
Bio-Rad/Propel



Labs


CytoFLEX
Beckman Coulter


FP 1000 Cell Preparation System
Beckman Coulter


Vi-CELL ® XR Cell Viability Analyzer
Beckman Coulter


FC 500 Series
Beckman Coulter


MoFlo ® Astrios TM
Beckman Coulter


Coulter Epics XL TM and XL-MCL TM
Beckman Coulter


Gallios TM
Beckman Coulter


CyAn TM ADP Analyzer
Beckman Coulter


Attune TM Acoustic Focusing Cytometer
Life Technologies


Attune ® NxT Acoustic Focusing Cytometer
Life Technologies


EVOS
Life Technologies


Countess II FL
Life Technologies


EC800 Cell Analyzer
Sony


SH800 Cell Sorter
Sony


SP6800 Spectral Analyzer
Sony


SY3200 Cell Sorter
Sony


A50-Micro'
Apogee Flow



Systems


A50-Universal
Apogee Flow



Systems


Auto40
Apogee Flow



Systems


FlowSight
Amnis


ImageStreamX Mark II
Amnis


JSAN
Bay Bioscience


CytoSense
CytoBuoy


CytoSub
CytoBuoy


CytoSense
CytoBuoy


CytoBuoy
CytoBuoy


Cytonome Viva TM G1
CYTONOME


GigaSort TM
CYTONOME


Hydris
CYTONOME


Agilent 2100 Bioanalyzer
Agilent



Technologies


NovoCyte
ACEA Biosciences


CyFlow ® Space
Partec technology


CyFlow ® Cube 8
Partec technology


CyFlow ® Cube 6
Partec technology


CyFlow ® Ploidy Analyser
Partec technology


CyFlow ® Counter
Partec technology


CyFlow ® miniPOC
Partec technology


CyFlow ® SL
Partec technology


CyFlow ® Sorter
Partec technology


CyFlow ® CCA
Partec technology


CyFlow ® Oenolyser
Partec technology


NucleoCounter ® NC-3000 TM
Chemometec


NucleoCounter ® NC-250 TM
Chemometec


NucleoCounter ® NC-200 TM - High Precision Cell
Chemometec


Counter


HPC-100 Portable Flow Cytometer
Cronus



Technologies Ltd


Cytell Cell Imaging System
GE Healthcare


MAGPIX
Luminex


Luminex ® 100/200 TM System
Luminex


FLEXMAP 3D ®
Luminex


ImageXpress ® Velos Laser Scanning Cytometer
molecular devices


ClonePix TM 2
molecular devices


SpectraMax ® i3
molecular devices


AQ1 Discrete Analyzer
SEAL Analytical



Ltd.


AQ2 Discrete Analyzer
SEAL Analytical



Ltd.


AQ400 Discrete Analyzer
SEAL Analytical



Ltd.


AQUA 900
SEAL Analytical



Ltd.


AA3 HR AutoAnalyzer
SEAL Analytical



Ltd.


AA1 AutoAnalyzer
SEAL Analytical



Ltd.


QuAAtro39
SEAL Analytical



Ltd.


Infralyzer 2000
SEAL Analytical



Ltd.


Technicon AutoAnalyzer II (AAII)
SEAL Analytical



Ltd.


Technicon/Bran + Luebbe TrAAcs 800-2000
SEAL Analytical



Ltd.


Bran + Luebbe FIA Analyzer
SEAL Analytical



Ltd.


BioSorter ® Large Particle Flow Cytometer
Union Biometrica,



Inc.


COPAS TM Large Particle Flow Cytometers
Union Biometrica,



Inc.


Cellometer Mini Cell Counter
Nexcelom


Cellometer Auto T4 Cell Viability Counter
Nexcelom


Cellometer Auto X4 Cell Viability Counter
Nexcelom


Cellometer Auto 1000 Cell Viability Counter
Nexcelom


Cellometer Auto 2000 Cell Viability Counter
Nexcelom


Cellometer Vision CBA
Nexcelom


Celigo S
Nexcelom


NovoCyte TM 1000
ACEA


NovoCyte TM 2000
ACEA


NovoCyte TM 2060
ACEA


NovoCyte TM 3000
ACEA


HPC-100
Handyem


S1000EXi
Stratedigm


SE520Xi
Stratedigm


Sysmex ® DI-60
Sysmex


CellaVision ® DM96
Sysmex


CellaVision ® DM1200
Sysmex


Cytation
BioTek


EasyCell Assistant
Medica


IN Cell Analyzer
GE Healthcare







Fluorish List








Big Blue
BD Biosciences


Kermit
Miltenyi


ac6
BD Biosciences


srDAs
BD Biosciences


a
BD Biosciences


FACSCanto II Immunology
BD Biosciences


Test Cyt
Millipore


milt
Miltenyi


ac
BD Biosciences


ietest
BD Biosciences


Curiel's Aria
BD Biosciences


AttuneÂ ® Acoustic Focusing Cytometer
Life Technologies


Blue/Violet


Medawar LSRII
BD Biosciences


Medawar Calibur
BD Biosciences


FACSAria INER
BD Biosciences


Attune R/A
Life Technologies


Fortessa
BD Biosciences


Aria
BD Biosciences


SORTER
BD Biosciences


Cyan
Beckman Coulter


LSR II
BD Biosciences


ARIA
BD Biosciences


Canto II
BD Biosciences


F09 - LSR Fortessa 1
BD Biosciences


“The Hoff”
BD Biosciences


6th Floor Hess Fortessa A
BD Biosciences


Cerebro BDFACSAriaII
BD Biosciences


Mystique BDFACSArialII
BD Biosciences


Godzilla BDFACSAriaII
BD Biosciences


Wolverine BDFACSAriaII
BD Biosciences


Megatron BDFACSAriaII
BD Biosciences


Megatron BDFACSAriaII
BD Biosciences


Fortessa B
BD Biosciences


6 colour Canto II
BD Biosciences


10 colour LSR II
BD Biosciences


4 laser 13 colour Influx sorter
BD Biosciences


14 colour X20
BD Biosciences


SORP
BD Biosciences


FACSAria INER
BD Biosciences


LSR561
BD Biosciences


Fortessa FCF UZH
BD Biosciences


LSR 2 B
BD Biosciences


LSRII-C
BD Biosciences


Cal 3
BD Biosciences


Aria II A
BD Biosciences


LSR 16
BD Biosciences


LSB Fortessa
BD Biosciences


IMMUN LSRII
BD Biosciences


IRC
BD Biosciences


UV LSR
BD Biosciences


5 Laser Aria
BD Biosciences


Curiel's LSR II
BD Biosciences


LSR Fortessa
BD Biosciences


Mauzeroll Aria
BD Biosciences


Frenette
BD Biosciences


Fallon
Beckman Coulter


Galios
Beckman Coulter


LSRIIFortessa
BD Biosciences


FACSCanto II CLSB
BD Biosciences


LSR II SC
BD Biosciences


UNCA Fortessa
BD Biosciences


VERSE
BD Biosciences


ARIAII
BD Biosciences


ARIAIII
BD Biosciences


F09 - BD LSRFortessa
BD Biosciences


HMRI FACSCanto II A
BD Biosciences


HMRI FACSCantoII B (HTS)
BD Biosciences


HMRI Aria III
BD Biosciences


L2
BD Biosciences


UoN Canto
BD Biosciences


LSRII M902
BD Biosciences


Fortessa 1
BD Biosciences


F05 - FACSAria
BD Biosciences


F02 - FACSAria III
BD Biosciences


F10 - BD FACSAria III
BD Biosciences


F03 - Guava
Millipore


Aria Blue 11 Color
BD Biosciences


Aria Red
BD Biosciences


Aria Orange
BD Biosciences


Aria Cyan
BD Biosciences


Aria Emerald
BD Biosciences


Aria Silver BSL3
BD Biosciences


LSR Fortessa
BD Biosciences


LSR II Bldg 4
BD Biosciences


LSR Fortessa bldg 4
BD Biosciences


CANTO II Bldg 50
BD Biosciences


4 Laser LSR II
BD Biosciences


5 Laser LSR II
BD Biosciences


FACSArray BL-2
BD Biosciences


FACSCalibur
BD Biosciences


DUAL for long term studies
BD Biosciences


MoFlo 1095 Production only
Beckman Coulter


BL-2 FACSAria III sorter
BD Biosciences


Astrios BL-2 sorter
Beckman Coulter


Tessy
BD Biosciences


LSR II-1
BD Biosciences


Fortessa
BD Biosciences


4 laser AriaIII
BD Biosciences


LSRFortessa
BD Biosciences


UoN FACSAria II cell sorter
BD Biosciences


Door
Beckman Coulter


Fortessa
BD Biosciences


WCI - FACSAria I
BD Biosciences


LSRII Karp8
BD Biosciences


Karp 8
BD Biosciences


Canto
BD Biosciences


Aria sorter
BD Biosciences


DI lab
BD Biosciences


DI FACSAria
BD Biosciences


Constance
BD Biosciences


DI FACSAria III
BD Biosciences


WCI_FACS Canto
BD Biosciences


MACSQuant 10
Miltenyi


VAMC Memphis LSR
BD Biosciences


VAMC Memphis S3
Bio-Rad


ARIA INER
BD Biosciences


Uhura
BD Biosciences


Kirk
BD Biosciences


Data
Millipore


Spock
BD Biosciences


McCoy
BD Biosciences









EXAMPLES
The present invention is further illustrated by reference to the following Examples. However, it should be noted that these Examples, like the embodiments described above, are illustrative and are not to be construed as restricting the scope of the invention in any way.
Example 1: Generation of Hydrogel Particles
Photomasks for UV lithography were sourced from CADart Services Inc. and were designed using AutoCad (AutoDesk, Inc.). SU-8 photo resist (Microchem, Inc.) was photo crosslinked on 4′′ silicon wafers using a collimated UV light source (OAI, Inc.) to create masters for microfluidic device fabrication. PDMS (polydimethylsiloxane, Sigma Aldrich, Inc.) was prepared and formed using standard published methods for soft lithography and microfluidic device fabrication (See, McDonald J C, et al., 2000, Electrophoresis 21:27-40).
Droplets were formed using flow-focusing geometry where two oil channels focus a central stream of aqueous monomer solution to break off droplets in a water-in-oil emulsion. A fluorocarbon-oil (Novec 7500 3M, Inc.) was used as the outer, continuous phase liquid for droplet formation. To stabilize droplets before polymerization, a surfactant was added at 0.5% w/w to the oil phase (ammonium carboxylate salt of Krytox 157 FSH, Dupont). To make the basic polyacrylamide gel particle, a central phase of an aqueous monomer solution containing N-acrylamide (1-20% w/v), a cross-linker (N,N′-bisacrylamide, 0.05-1% w/v), an accelerator, and ammonium persulfate (1% w/v) was used. An accelerator, (N,N,N′,N′tetramethylethylenediamine (2% vol %) was added to the oil-phase in order to trigger hydrogel particle polymerization after droplet formation.
Several co-monomers were added to the basic gel formulation to add functionality. Allyl-amine provided primary amine groups for secondary labeling after gel formation. We modulated forward scatter by adjusting the refractive index of the gel by adding co-monomers allyl acrylate and allyl methacrylate. Side scattering of the droplets was tuned by adding a colloidal suspension of silica nanoparticles and/or PMMA (poly(methyl methacrylate)) particles ( ̃100 nm) to the central aqueous phase prior to polymerization.
Stoichiometric multiplexing of the hydrogel particles was achieved by utilizing co-monomers containing chemically orthogonal side groups (amine, carboxyl, maleimide, epoxide, alkyne, etc.) for secondary labeling.
Droplets were formed at an average rate of 5 kHz and were collected in the fluorocarbon oil phase. Polymerization was completed at 50° C. for 30 minutes, and the resulting hydrogel particles were washed from the oil into an aqueous solution.
Example 2: Generation and Visualization of 12 11 m Hydrogel Particles
Water containing 5% acrylamide, 0.25% bisacrylamide, 0.05% allyl amine, and 0.1% ammonium persulfate was flowed through a center channel and focused by oil containing 0.1% TEMED through a 10 micron nozzle to produce 10 μm hydrogel particles, shown in FIG. 3A. Following polymerization, the particles were washed in water, shown in FIG. 3B, and conjugated to dyes of interest. The fluorescent hydrogel particles were visualized with fluorescence microscopy, shown in FIG. 3C.
Example 3: Multidimensional Tuning of Hydrogel Particle Optical Properties
As depicted in FIG. 4, hydrogel particles are tuned in multiple dimensions to match specific cell types unlike polystyrene beads. Cells are deconvolved using combinations of optical parameters such as FSC and SSC (FIG. 4A) or secondary markers. Hydrogel particles are tuned to match the SSC and FSC of specific cell types unlike polystyrene beads (brown) which are limited in size (FSC) and side scattering (FIG. 4B). Hydrogel particles are further functionalized with stoichiometrically tuned ratios of specific chemical side-groups and secondary labels allowing the cell type to be precisely matched without suffering from biological noise as fixed cell lines do (FIG. 4C).
Example 4: Flow Cytometer Delay Time as a Function of Hydrogel Particle Diameter
As shown in FIG. 5, the inter-drop delay for a flow cytometer can be precisely correlated to hydrogel particle diameter. Data are shown for hydrogel particles of 3, 6, 10, 32, and 50 μm diameters using flow cytometer nozzle sizes of 70 and 100 μm.
Example 5: Comparison of Hydrogel Particles with Encapsulated DNA to Cells
To form hydrogel particles with encapsulated DNA, 40 μg/mL-1000) μg/mL of reconstituted calf thymus DNA was added to a polymer mix containing 20% 19:1 (acrylamide:bis-acrylamide) and 0.1% allyl amine in water. 0.4% ammoniumpersulfate was added to the mix prior to droplet formation. Hydrogel particles were formed as described in Example 1. Hydrogel particles with 200 μg/mL of encapsulated calf thymus DNA displayed cell-like staining using propidium iodide as visualized using a commercial imaging cytometer and compared to Chinese Hamster Ovary cells stained using the same procedure. Images were obtained using a Nexcelom CellometerTM (FIG. 6).
Cells obtained from a buccal swab were washed in PBS and stained with propidium iodide. In parallel, populations of hydrogel particles containing a range of DNA concentrations were also stained in the same manner. Both the cell and particle suspensions were analyzed on a flow cytometer (488/590 nm excitation/emission). Flow cytometry analysis of cheek cells and the same range of encapsulated DNA particles showed that the particles display a range of cell-like fluorescent properties (FIG. 7, left panel). The intensity of staining shows a linear correlation with the median intensity as measured by flow cytometry (FIG. 7, right panel).
Example 6: Tuning of Hydrogel Particle Side Scattering
Colloidal silica was added at 12.5%, 6.25%, 3.125% and 0% to the aqueous fraction of the polymer mix and hydrogel particles were formed as described in Example 1. Forward and side scattering data were obtained using a flow cytometer. The results showed that side scatter signal (FIG. 8, left panel) increased with higher percentages of encapsulated nanoparticles while forward scatter (FIG. 8, right panel) remained generally unchanged, demonstrating the independent tuning of side scatter and forward scatter.
Example 7: Tuning of Hydrogel Particle Forward Scattering
In this experiment, the percentage of acrylamide:bis-acrylamide in the hydrogel composition was varied from between 10 and 40% to tune the refractive index of the hydrogel particles as measured by forward scattering in a flow cytometer. As shown in FIG. 9, the forward scattering increased with increasing percentages of acrylamide:bisacrylamide as a fraction of water.
Example 8: Tuning of Hydrogel Particle Optical Properties
An example of tuning hydrogel particles to match optical properties of a desired cell subtype. Co/monomers can be combined with nanoparticles to tune both forward and side scatter properties of the hydrogels using passive optical measurements in a flow cytometer. By combining these properties with chemically labile co-monomers (e.g. allyl amine, acrylic acid), additional fluorophores/proteins/biological side groups can be added and labeled (if desired) in order to match cell subpopulation staining in addition to scattering properties. These are the three primary metric by which cells are identified using flow cytometry. Additional side groups, such as those containing heavy metals, can be used for Cy-TOF (cytometry, time of flight mass spectrometry) calibration for example. Finally, biocompatible material can be encapsulated to mimic subcellular organelle staining.
Example 9: Tuning of Hydrogel Particle Optical Properties
A 50 nm nanoparticle colloidal suspension was incorporated into the hydrogel matrix to mimic the optical properties of lymphocytes and monocytes (FIGS. 13A and 13B). The percent composition of the suspension was altered to match the blood cell subpopulations from the blood sample control (Streck) (FIG. 13C).
Specifically, the concentration of the acrylamide monomer (0.7-0.8M) of the hydrogel particle was adjusted to increase the forward scatter of the particles to match blood cell subpopulations. The percentage of bisacrylamide cross linker can also be changed to affect forward scatter (1-5%). Silica nanoparticles were used at 5% or 10% in the compositions to adjust side scatter. The results of this experiment are shown in FIG. 13.
All, documents, patents, patent applications, publications, product descriptions, and protocols which are cited throughout this application are incorporated herein by reference in their entireties for all purposes.
The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art the best way known to the inventors to make and use the invention. Modifications and variation of the above-described embodiments of the invention are possible without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. It is therefore understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described.

## Claims

1. A method for determining if a sample includes one or more of lymphocytes and monocytes, comprising:
inserting, into a cytometric device, a plurality of hydrogel particles suspended in an aqueous solution, the plurality of hydrogel particles including: (1) a first portion of hydrogel particles having forward scatter, side scatter, and hydrodynamic properties substantially similar to a lymphocyte, and (2) a second portion of hydrogel particles having forward scatter, side scatter, and hydrodynamic properties substantially similar to a monocyte, wherein each hydrogel particle of the plurality of hydrogel particles comprises a polymerized monomer and has at least one functionalized surface having at least one cell surface marker;
measuring the forward scatter, side scatter, and hydrodynamic properties of each hydrogel particle of the plurality of hydrogel particles using the cytometric device;
calibrating the cytometric device based on the measured forward scatter, side scatter, and hydrodynamic properties of the plurality of hydrogel particles;
adding a sample comprising a plurality of cells to the calibrated cytometric device; and
measuring the forward scatter, side scatter, and hydrodynamic properties of each cell of the plurality of cells using the calibrated cytometric device to determine if the sample includes one or more of lymphocytes and monocytes.

2. A method for detecting a white blood cell in a sample, comprising:
inserting, into a cytometric device, a hydrogel particle in aqueous solution, the hydrogel particle having optical-scatter and hydrodynamic properties substantially similar to a white blood cell, the hydrogel particle comprising a polymerized monomer and being functionalized such that it includes at least one cell surface marker;
measuring the optical-scatter and hydrodynamic properties of the hydrogel particle using the cytometric device to calibrate the cytometric device;
inserting a sample in the cytometric device, the sample comprising a plurality of cells;
measuring the optical-scatter and hydrodynamic properties of individual cells of the plurality of cells; and
determining, based on the measurements of the optical-scatter and hydrodynamic properties, whether a white blood cell or plurality thereof is present in the sample.

3. The method of claim 2, further comprising sorting the white blood cell or plurality of white blood cells to a separate vessel if one or more white blood cells is determined to be present in the sample.

4. The method of claim 2, further comprising: inserting a plurality of hydrogel particles into the cytometric device, wherein each hydrogel particle of the plurality of hydrogel particles has at least one optical-scatter or hydrodynamic property substantially similar to a white blood cell sub-type, wherein the cytometric device is calibrated based on measured optical-scatter and hydrodynamic properties of each hydrogel particle of the plurality of hydrogel particles.

5. The method of claim 4, wherein at least one optical-scatter property is the same for each hydrogel particle of the plurality of hydrogel particles.

6. The method of claim 2, wherein at least one optical-scatter property is side scatter (SSC).

7. The method of claim 2, wherein at least one optical-scatter property is forward scatter (FSC).

8. The method of claim 2, wherein the hydrogel particle has a fluorescence emission substantially similar to a white blood cell, the method further comprising:
measuring the fluorescence emission of the hydrogel particle using the cytometric device to calibrate the cytometric device; and
measuring the fluorescence emission of individual cells of the plurality of cells, wherein determining whether a white blood cell or plurality thereof is present in the sample is further based on measured fluorescence emissions.

9. The method of claim 2, wherein at least one optical-scatter property is a ratio of FSC to SSC.

10. The method of claim 2, wherein the polymerized monomer of the hydrogel particle is selected from hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glycerol methacrylate (GMA), glycol methacrylate, ethylene glycol, fumaric acid, 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, poly(ethylene glycol) methacrylate, methoxy-poly(ethylene glycol) methacrylate, methacrylic acid, sodium methacrylate, glycerol methacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate, phenyl acrylate, phenyl methacrylate, benzyl acrylate, benzyl methacrylate, 2-phenylethyl acrylate, 2-phenylethyl methacrylate, 2-phenoxyethyl acrylate, 2-phenoxyethyl methacrylate, phenylthioethyl acrylate, phenylthioethyl methacrylate, 2,4,6-tribromophenyl acrylate, 2,4,6-tribromophenyl methacrylate, pentabromophenyl acrylate, pentabromophenyl methacrylate, pentachlorophenyl acrylate, pentachlorophenyl methacrylate, 2,3-dibromopropyl acrylate, 2,3-dibromopropyl methacrylate, 2-naphthyl acrylate, 2-naphthyl methacrylate, 4-methoxybenzyl acrylate, 4-methoxybenzyl methacrylate, 2-benzyloxyethyl acrylate, 2-benzyloxyethyl methacrylate, 4-chlorophenoxyethyl acrylate, 4-chlorophenoxyethyl methacrylate, 2-phenoxyethoxyethyl acrylate, 2-phenoxyethoxyethyl methacrylate, N-phenyl acrylamide, N-phenyl methacrylamide, N-benzyl acrylamide, N-benzyl methacrylamide, N,N-dibenzyl acrylamide, N,N-dibenzyl methacrylamide, N-diphenylmethyl acrylamide N-(4-methylphenyl)methyl acrylamide, N-1-naphthyl acrylamide, N-4-nitrophenyl acrylamide, N-(2-phenylethyl)acrylamide, N-triphenylmethyl acrylamide, N-(4-hydroxyphenyl)acrylamide, N,N-methylphenyl acrylamide, N,N-phenyl phenylethyl acrylamide, N-diphenylmethyl methacrylamide, N-(4-methyl phenyl)methyl methacrylamide, N-1-naphthyl methacrylamide, N-4-nitrophenyl methacrylamide, N-(2-phenylethyl)methacrylamide, N-triphenylmethyl methacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenyl methacrylamide, N,N′-phenyl phenylethyl methacrylamide, N-vinyl carbazole, 4-vinylpyridine, 2-vinylpyridine, or a combination thereof.

11. The method of claim 2, wherein the polymerized monomer of the hydrogel particle is a biodegradable monomer.

12. The method of claim 11, wherein the biodegradable monomer is a monosaccharide, disaccharide, polysaccharide, peptide, protein, or protein domain.

13. The method of claim 12, wherein the biodegradable monomer is a protein or protein domain comprising at least one non-natural amino acid.

14. The method of claim 11, wherein the biodegradable monomer is a structural polysaccharide.

15. The method of claim 11, wherein the biodegradable monomer is agar, agarose, alginic acid, alguronic acid, alpha glucan, amylopectin, amylose, arabinoxylan, beta-glucan, callose, capsullan, carrageenan polysaccharide, cellodextrin, cellulin, cellulose, chitin, chitosan, chrysolaminarin, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, dextran, ficoll, fructan, fucoidan, galactoglucomannan, galactomannan, galactosaminoogalactan, gellan gum, glucan, glucomannan, glucorunoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hypromellose, icodextrin, inulin, kefiran, laminarin, lentinan, levan polysaccharide, lichenin, mannan, mixed-linkage gluxan, paramylon, pectic acid, pectin, pentastarch, phytoglycogen, pleuran, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, sinistrin, sizofiran, welan gum, xanthan gum, xylan, xyloglucan, zymosan, or a combination thereof.

16. The method of claim 11, wherein the biodegradable monomer is chitosan or hyaluronan.

17. The method of claim 12, wherein the protein is a structural protein, a domain thereof, or a combination thereof.

18. The method of claim 12, wherein the protein is a proteoglycan, a domain thereof, or a combination thereof.

19. The method of claim 12, wherein the protein is an extracellular matrix component.

20. The method of claim 18, wherein the proteoglycan is decorin, biglycan, testican, bikunin, fibromodulin, lumican, a domain thereof, or a combination thereof.

21. The method of claim 19, wherein the protein is elastin or a proteoglycan.

22. The method of claim 19, wherein the protein is collagen.

23. The method of claim 22, wherein the collagen is collagen type I, collagen type II, collagen type III, a domain thereof or a combination thereof.

24. The method of claim 2, wherein the polymerized monomer of the hydrogel particle is bifunctional.

25. The method of claim 2, wherein the at least one cell surface marker is one of the cell surface markers set forth in Table 4, or a combination thereof.

26. A method for calibrating a cytometric device for analysis of white blood cells, comprising:
inserting, into a cytometric device, a plurality of hydrogel particles suspended in an aqueous solution, each hydrogel particle of the plurality of hydrogel particles having side scatter, forward scatter, and hydrodynamic properties substantially similar to one of a lymphocyte, monocyte, or granulocyte, each hydrogel particle comprising a polymerized monomer and configured with at least one functionalized surface having at least one cell surface marker;
measuring the side scatter, forward scatter, and hydrodynamic properties of each hydrogel particle of the plurality of hydrogel particles using the cytometric device; and
calibrating the cytometric device for analysis of white blood cells based on the measured side scatter, forward scatter, and hydrodynamic properties of the plurality of hydrogel particles.