bluuebunny/crossref_metadata_2025 huggingface dataset:
Measurement of In Vitro Specific‐Antibody Synthesis
Authors: Yarchoan Robert
Published: June 2004

In this unit, a protocol is provided in which antigen‐specific responses by human lymphocytes are elicited and then quantitated by the enzyme‐linked immunosorbent assay (ELISA). Initially, Ficoll‐Hypaque‐isolated mononuclear cells are treated to remove cytophilic antibody. The antibody response being studied is then elicited by the appropriate stimulating antigen (in this case, influenza virus, although other antigens can also be used) or mitogen. Finally, after a 12‐day incubation, the supernatant is harvested for assay.
Measurement of Polyclonal and Antigen‐Specific Cytotoxic T Cell Function
Authors: Biddison William E. , Lichtenfels Rudolf , Adibzadeh Medi , Martin Roland
Published: June 2004

Measurement of in vitro cytotoxic function of human T cells can be accomplished by polyclonal stimulation of T cell effectors using anti‐CD3 antibody, which stimulates all cytolytic effector cells, or with a specific stimulating antigen. Accordingly, two sets of assays of cytolytic T cell function are described in this unit, one for measuring anti‐CD3‐mediated cytotoxicity and the other for measuring antigen‐specific cytotoxicity. Although the calcein release assay (CARE‐LASS) described here is for use with antigen‐activated cytotoxic T lymphocytes (CTL) as well as natural killer (NK) or lymphokine‐activated killer (LAK) cells, minor changes in the protocols that address polyclonal T cell activation are described that make them suitable for use with calcein‐labeled target cells.
Measurement of Cytotoxic Activity of NK/LAK Cells
Authors: Whiteside Theresa L.
Published: June 2004

In this unit, methodology is presented for measuring the capacity of human natural killer (NK) or lymphokine‐activated killer (LAK) cells to lyse tumor cell targets. Cytotoxicity of these effector cells is evaluated in a short‐term 51Cr‐release assay using NK‐sensitive tumor cells as the targets for NK cells or NK‐resistant tumor cells as the targets for LAK cells. In the basic protocol, a generic 51Cr‐release assay is described in which PBMC, purified NK cells or interleukin 2 (IL‐2)‐activated lymphocyte populations (LAK cells) are utilized as effector cells. Support protocols describe preparation of nonadherent tumor cells, cells obtained from malignant effusions, trypsinized tumor cells from adherent monolayer culture, or freshly isolated tumor cells from surgical specimens. All of these cell types can serve as the 51Cr‐labeled target cells in the basic protocol. The procedure for labeling target cells from any of these sources with 51Cr is also provided.
Generation and Maintenance of Cloned Human T Cell Lines
Authors: Yssel Hans , Spits Hergen
Published: June 2004

This unit describes protocols for the generation of human (allo‐) antigen‐specific T cell lines, and T lymphocyte subpopulations with distinct cytokine production profiles from purified peripheral or cord blood CD4+ T cells, respectively. Methods for the cloning and maintenance of these cell lines are given, as well as a protocol for freeze/thaw procedures.
Generation of T Cell Hybridomas
Authors: Mayer Lloyd
Published: June 2004

This unit presents a method of immortalizing T cells by fusing them with a preexisting T cell line (the fusion partner) to form a T cell hybridoma. Support protocols present key elements in successful fusions, including the selection of an appropriate fusion partner and activation of the donor T cells. Other support protocols describe the selection processes used for hybridomas, including measures to ensure long‐term stability and maintenance of cell lines.
Generation of Epstein‐Barr Virus (EBV)–Immortalized B Cell Lines
Authors: Tosato Giovanna
Published: June 2004

Immortalization of B lymphocytes by EBV is an effective procedure for inducing long‐term growth of certain human B lymphocytes. The protocol described in this unit to accomplish this can be divided into three stages: preparation of virus, preparation of target cells to be immortalized, and EBV infection and growth of infected cells.
Isolation of Tissue Mast Cells
Authors: Metcalfe Dean D.
Published: June 2004

Mast cells, unlike other immune effector cells, are not found in the circulation and thus must be obtained from accessible tissues such as gastrointestinal and lung tissue. In this unit, a combination of mechanical fragmentation, enzyme digestion, and centrifugation procedures are used to obtain suspensions enriched for mast cells from gastrointestinal tissue. A support protocol details a procedure for acid toluidine blue staining of mast cells.
Isolation and Characterization of Human Natural Killer Cell Subsets
Authors: Cooper Megan A. , Caligiuri Michael A.
Published: June 2004

Natural killer (NK) cells are innate immune lymphocytes that play a critical role in the host defense against pathogens through their production of cytokines and cytotoxic activity. Human NK cells can be divided into two subsets, each with distinct phenotypic and functional properties, based on cell‐surface density expression of CD56 (CD56bright and CD56dim). This unit describes the purification of human NK cell subsets from blood, protocols for the further characterization of NK cell function, and further background information on these cell types.
Subcellular Fractionation of Tissue Culture Cells
Authors: Aniento Fernando , Gruenberg Jean
Published: June 2004

Development of cell fractionation techniques over the last few decades has provided the means to analyze the composition and properties of purified cellular elements. In particular, subcellular fractionation is essential for the development of cell‐free assays that reconstitute complicated cellular processes. These assays have provided new and important tools to understand the molecular mechanisms of complex cellular functions, permitting these functions to be studied in the test tube as a series of biochemical reactions. The protocols in this unit describe fractionation of tissue culture cells to immunoisolate early and late endosomes under conditions where these compartments retain their capacity to support membrane transport in vitro.
Immunoprecipitation
Authors: Bonifacino Juan S. , Dell'Angelica Esteban C. , Springer Timothy A.
Published: June 2004

Immunoprecipitation consists of multiple ordered steps: lysing the cell with detergent if the antigen (usually a protein) to be precipitated is membrane‐bound; binding of a specific antigen to an antibody; precipitating the antibody‐antigen complex; washing the precipitate; and dissociating the antigen from the immune complex. The dissociated antigen is then analyzed by electrophoretic methods. In this unit, the basic protocol details the immunoprecipitation of a radiolabeled antigen with a specific antibody (polyclonal or monoclonal) covalently linked to Sepharose. Preparation of Ab‐Sepharose is described in the Support Protocol. The first two alternate protocols present methods for precipitating or isolating the soluble immune complexes formed between a specific antibody and a radiolabeled antigen. Immunoprecipitation is achieved with polyclonal anti‐immunoglobulin (Ig) serum, anti‐Ig‐Sepharose, Staphylococcus protein A or Streptococcus protein G bound to Sepharose, or Staphylococcus aureus bacteria which contain protein A on the cell surface. The third alternate protocol should be used for immunoprecipitation of antigens that are nonspecifically associated with other proteins. The fourth alternate protocol describes immunoprecipitation of unlabeled protein antigens with Ab‐Sepharose.
One‐Dimensional Gel Electrophoresis of Proteins
Authors: Gallagher Sean R. , Smith John A.
Published: June 2004

This unit provides several methods for one‐dimensional gel electrophoresis of proteins. The basic protocol is the standard method used for gel electrophoresis under denaturing conditions. The first alternate protocol provides conditions used for nondenaturing gel electrophoresis. Gradient gels are described and resolve a much wider molecular‐weight range of proteins on a single gel. Minigels, both single‐concentration and gradient, are also described.
Isolation of Proteins for Microsequence Analysis
Authors: Moos Malcolm
Published: June 2004

The first basic protocol in this unit can be used to determine the amino‐terminal sequence of polypeptides or proteins. It is particularly appropriate for large fragments of insoluble or hydrophobic proteins or proteins that cannot be purified to >90% molar purity without electrophoresis. Although the efficacy of this technique varies with the protein, it is possible to obtain useful sequence information starting with less than 50 pmol of the protein of interest. If the protein is blocked at the amino terminus, chemical cleavage or partial enzymatic digestion must be performed prior to electrophoresis. Upon isolation, the internal amino acid sequence is analyzed as described in the second basic protocol. This method requires ˜200 pmol of protein for analysis.
Staining Proteins in Gels
Authors: Sasse Joachim , Gallagher Sean R.
Published: June 2004

This unit describes protocols for detecting protein in a gel by either Coomassie blue, silver or fluorescent staining. Alternate rapid staining procedures are provided for each method and a support protocol describes how to photograph stained gels. Fluorescent staining (e.g., with SYPRO Orange or Red) is described as a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and often as sensitive as silver staining. Staining of proteins in SDS‐polyacrylamide gels is described, and an alternate protocol details variations in the procedure for proteins in nondenaturing gels. A final support protocol describes the photography of fluorescently stained proteins.
Iodination of Soluble and Membrane‐Bound Proteins
Authors: Samelson L.E.
Published: June 2004

Coupling of radioactive 125I to soluble proteins is a critical step in a number of immunoassays. Chloramine T‐catalyzed iodination of soluble proteins, such as immunoglobulins, is described in this unit. Protocols using lactoperoxidase‐catalyzed iodination of soluble or membrane‐bound proteins are also described.
Biosynthetic Labeling of Proteins
Authors: Bonifacino Juan S.
Published: June 2004

Biosynthetic labeling techniques are commonly used in the study of biochemical properties, synthesis, processing, intracellular transport, secretion, and degradation of proteins. In this unit, protocols are described for biosynthetically labeling many secreted and membrane proteins. The describes short‐term labeling of cells in suspension (30 min to 3 hr) and an alternate protocol describes a modification of this procedure to be used for adherent cells. Alternate protocols for long‐term (3 to 24 hr) and pulse‐chase labeling of cells are also presented. Label incorporation can be determined as described in a support protocol. The cells used in these protocols can be either from suspension cultures or from single‐cell suspensions of spleen or thymus.
Metabolic Radiolabeling of Animal Cell Glycoconjugates
Authors: Diaz Sandra , Varki Ajit
Published: June 2004

This unit describes metabolic labeling techniques that provide specific information about the structure, sequence, and distribution of the sugar chains of glycoconjugates. In the Basic Protocol, cells in culture are grown through several population doublings in complete medium supplemented with radiolabeled glycoconjugate precursors to reach a steady‐state level of incorporation. In the alternate protocols, cells are cultured for a short period of time in a deficient medium that contains a high concentration of radiolabeled precursor. A pulse or pulse‐chase labeling procedure is provided to analyze precursor‐product relationships. With the sequential pulse‐labeling method described here, it is possible to obtain quantities of labeled glycoconjugates with the use of a minimum amount of labeled precursor by using the same batch of medium to pulse‐label a series of cultures. A support protocol describes the preparation of multiply deficient medium (MDM) for use in making appropriate deficient media.
Inhibition of N‐Linked Glycosylation
Authors: Powell Leland D.
Published: June 2004

Treatment of cells with inhibitors of the enzymes that synthesize N‐linked oligosaccharide chains results in production of glycoproteins containing missing or altered chains. This approach is useful for examining potential functional role(s) of this class of oligosaccharides on specific proteins or intact cells. This unit describes the use of inhibitors to prevent N‐linked glycosylation of proteins in cultured cells. First, the optimal concentration of inhibitor for the experiment (i.e., highest nontoxic concentration) is determined by monitoring [35S]methionine incorporation as a measure of protein biosynthesis. The ability of the inhibitor to hinder oligosaccharide processing is then determined by analyzing cells labeled with [3H]mannose using TCA precipitation or endo H digestion. Further suggestions are given on how to use methods for identifying a specific glycoprotein (if available) to measure the effect of the inhibitor on its N‐linked oligosaccharide chains. A Support Protocol details a method for concentrating proteins by acetone precipitation.
Production of Anti‐Peptide Antisera
Authors: Carter John Mark
Published: June 2004

The protocols described in this unit can be used for generating and testing anti‐peptide antisera, assuming that a scientist has already selected and conjugated peptides for experimental vaccines. Described are immunization and bleeding of experimental animals, a simple ELISA for antiserum screening, and affinity purification of the serum, including preparation of columns.
Identification of B Cell and T Cell Epitopes Using Synthetic Peptide Combinatorial Libraries
Authors: Pinilla Clemencia , Appel Jon R. , Houghten Richard A.
Published: June 2004

This unit presents a combinatorial library method that consists of the synthesis and screening of mixture‐based synthetic combinatorial libraries (SCL) of peptide molecules. The protocols employ peptide SCL to identify peptides recognized by mAbs and T cells. The first protocol uses a positional scanning SCL (PS‐SCL) made up of hexapeptides to identify antigenic determinants recognized by mAbs. The 120 mixtures in the hexapeptide PS‐SCL are tested for their inhibitory activity in a competitive ELISA. The second protocol uses a decapeptide PS‐SCL to identify T cell peptide ligands. The 200 mixtures of the decapeptide PS‐SCL are tested for their ability to induce proliferation. Support protocols cover optimization of the assay conditions for each mAb or T cell, to achieve the best level of sensitivity and reproducibility, and preparation of a hexapeptide PS‐SCL, along with deconvolution approaches.
Agarose Gel Electrophoresis
Authors: Voytas Daniel
Published: June 2004

Agarose gel electrophoresis is a simple and highly effective method for separating, identifying, and purifying 0.5‐ to 25‐kb DNA fragments. The Basic Protocol in this unit can be divided into three stages: (1) a gel is prepared with an agarose concentration appropriate for the size of DNA fragments to be separated; (2) the DNA samples are loaded into the sample wells and the gel is run at a voltage and for a time period that will achieve optimal separation; and (3) the gel is stained or, if ethidium bromide has been incorporated into the gel and electrophoresis buffer, visualized directly upon illumination with UV light. Support protocols describe the use of midigels and minigels, as well as recommendations for photographing stained gels.
Isolation and Purification of Large DNA Restriction Fragments from Agarose Gels
Authors: Moore David , Chory Joanne , Ribaudo Randall K.
Published: June 2004

This unit describes methods for recovering and purifying DNA restriction fragments from agarose gels. The first basic protocol describes electroelution of the fragment of interest from standard agarose gels using buffer‐filled dialysis bags, followed by concentration and purification using an Elutip column. This approach can be used effectively for fragments of all sizes from 50 to 20,000 bp. Electrophoresis directly onto NA‐45 paper is also described and provides relatively high yields for fragments smaller than 2000 bp. Protocols are also provided for separating fragments larger than 1000 bp using low gelling/melting agarose gels and purified by phenol extraction, b‐agarase digestion of the gel, or via glass beads extraction. Sieving agarose gels can also be used to resolve very small DNA fragments. Removing linkers from a fragment using a column rather than a gel is included, followed by a method for estimating DNA concentrations in solution.
Southern Blotting
Authors: Brown Terry
Published: June 2004

Southern blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support, resulting in immobilization of the DNA fragments, so the membrane carries a semipermanent reproduction of the banding pattern of the gel. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified. This unit describes Southern blotting via upward capillary transfer of DNA from an agarose gel onto a nylon or nitrocellulose membrane, and subsequent immobilization by UV irradiation (for nylon) or baking (for nitrocellulose). A describes how to calibrate a UV transilluminator for optimal UV irradiation of a nylon membrane. An alternate protocol details transfer using nylon membranes and an alkaline buffer, and is primarily used with positively charged nylon membranes. A second alternate protocol describes a transfer method based on a different transfer‐stack setup. The traditional method of upward capillary transfer of DNA from gel to membrane has certain disadvantages, notably the fact that the gel can become crushed by the weighted filter papers and paper towels that are laid on top of it. This slows down the blotting process and may reduce the amount of DNA that can be transferred. The downward capillary method described in the second alternate protocol is therefore more rapid and can result in more complete transfer.
Digestion of DNA with Restriction Endonucleases
Authors: Bloch Kenneth D.
Published: June 2004

Restriction endonucleases recognize short DNA sequences and catalyze the cleavage of double‐stranded DNA at specific sites within or adjacent to the recognition sequences. This unit describes how to cleave DNA and refers the investigator to frequently updated manuals by endonuclease suppliers for the precise optimized conditions. Alternate procedures are given for digesting a DNA sample with several different enzymes and digesting multiple DNA samples with the same endonuclease.
Enzymatic Labeling of Nucleic Acids
Authors: Tabor Stanley , Boyle Ann
Published: June 2004

Extensive knowledge of the enzymology involved in biosynthesis and degradation of nucleic acids has permitted the development of simple methods for labeling RNA and DNA with radioisotopes or biotin. These labeled probes are used primarily for hybridization to nucleic acid fragments of interest in a variety of applications. A complete description of the methods available for such labeling is beyond the scope of this manual, but contained within this unit are protocols for oligonucleotide‐primed synthesis of radiolabeled and biotinylated DNA probes, in vitro synthesis of radiolabeled RNA, and end labeling of synthetic oligonucleotide probes.
Analysis of RNA by Northern and Slot‐Blot Hybridization
Authors: Brown Terry
Published: June 2004

Specific sequences in RNA preparations can be detected by blotting and hybridization analysis using techniques very similar to those originally developed for DNA. Fractionated RNA is transferred from an agarose gel to a membrane support (northern blotting); unfractionated RNA is immobilized by slot or dot blotting. The resulting blots are studied by hybridization analysis with labeled DNA or RNA probes. The in this unit describes blotting and hybridization of RNA fractionated in an agarose/formaldehyde gel. This is arguably the quickest and most reliable method for northern analysis of specific sequences in RNA extracted from eukaryotic cells. An alternate protocol gives details of the glyoxal/DMSO method for denaturing gel electrophoresis, which may provide better resolution of some RNA molecules. A second describes slot‐blot hybridization of RNA samples, a rapid method for assessing the relative abundance of an RNA species in extracts from different tissues.
Calcium Phosphate Transfection
Authors: Kingston Robert E. , Chen Claudia A. , Okayama Hiroto
Published: June 2004

This unit contains two methods of calcium phosphate‐based eukaryotic cell transfection, protocols that can be used for both transient and stable transfections. In the protocols, plasmid DNA is introduced to monolayer cell cultures via a precipitate that adheres to the cell surface. The uses a HEPES‐buffered solution to form a calcium phosphate precipitate that is directly layered onto the cells. In the alternate high‐efficiency method, a BES‐buffered system is used that allows the precipitate to form gradually in the medium and is then dropped onto the cells. The alternate method is particularly efficient for stable transformation of cells with circular plasmid DNA, and may be helpful with linear or genomic DNA. Both methods of transfection require very high‐quality plasmid DNA, which can be prepared as described in the second . Transfection efficiency in some cell lines can be increased by shocking the cells with glycerol or dimethyl sulfoxide (DMSO) as described in the first .
Transfection Using DEAE‐Dextran
Authors: Selden Richard F
Published: June 2004

Two protocols for DEAE‐dextran transfection of cells are provided in this unit. The describes a procedure used to transfect adherent cells and the first presents a method used to transfect suspension cells. If an increase in transfection efficiency is needed, cells can be treated with chloroquine as described in the second .
Use of Monoclonal Antibodies for Expression Cloning
Authors: Hollenbaugh Diane , Aruffo Alejandro , Jones Bryan , Linsley Peter
Published: June 2004

This unit details the use of transient expression in mammalian cells to screen cDNA libraries with monoclonal antibodies (MAb) to isolate cDNA clones encoding cell‐surface and intracellular proteins. The first protocol describes the cloning of cDNAs encoding cell‐surface antigens. The second protocol is a modification that facilitates isolation of cDNAs encoding antigens that are expressed intracellularly. Both protocols are designed for use with the expression vector CDM8, which contains a polylinker for subcloning double‐stranded cDNA.
Quantitation of Rare DNAs by PCR
Authors: Coen Donald M.
Published: June 2004

This unit presents a protocol that uses the polymerase chain reaction (PCR) to quantitate the numbers of a particular DNA sequence from 1 to 20,000 molecules per sample. In addition, it helps assess the presence of contaminating sequences, the bane of this kind of procedure. The DNA of interest is prepared and its concentration is determined. A known amount of this DNA is then mixed with two sets of oligonucleotide primers, one set specific for the DNA of interest (e.g., a virus) and the other set specific for an internal control (e.g., a single‐copy gene encoded by the host organism). The sequences between the primers are amplified, electrophoresed on a gel, transferred to a filter, and probed with oligonucleotides specific for each amplified product. The amounts of the amplified products from the DNA of interest can then be quantitated by comparison to the internal control. For simplicity, the protocol is written in terms of quantitating viral DNA molecules relative to host cellular sequences; however, it can be adapted readily for other applications.
Detection of Isotype Switch Rearrangement in Bulk Culture by PCR
Authors: Max Edward E. , Mills Frederick C. , Chu Charles
Published: June 2004

When a B lymphocyte changes from synthesizing IgM to synthesizing IgG, IgA, or IgE, this isotype switch is generally accompanied by a unique DNA rearrangement. The protocols in this unit describe two polymerase chain reaction (PCR)‐based strategies for detecting switch rearrangements in bulk culture. The first involves direct PCR across the switch junctions, providing the opportunity for characterizing the recombination products by nucleotide sequence analysis; however, because of characteristics inherent to the PCR methodology this strategy cannot easily be used as a quantitative assay for recombination. A support protocol details the preparation of the 5' Su PCR probe for this protocol. The second basic protocol describes a method known as digestion‐circularization PCR (DCPCR) that is more amenable to quantitation but yields no information on structure of the recombination products. Both techniques should be capable of detecting reciprocal deletion circles as well as functional recombination products remaining on the expressed chromosome.
Double‐Stranded DNA Sequence Analysis
Authors: Morrison Sherie L.
Published: June 2004

There are several different methods for analyzing the sequence of cloned DNA segments. For analysis of immunoglobulin variable (V) regions, double‐stranded DNA sequencing is routinely used. Although this method does not yield the longest DNA sequences, it is easy and provides sufficient information to determine the correct sequence. The protocols in this unit are for use with the Sequenase Version 2.0 kit from U.S. Biochemicals, which is based on dideoxy sequencing with a modified T7 DNA polymerase. The describes performing a sequencing reaction for cloned immunoglobulin V regions and running and analyzing a sequencing gel. A support protocol describes pouring and setting up the sequencing gel.
Spectratype/Immunoscope Analysis of the Expressed TCR Repertoire
Authors: Currier Jeffrey R. , Robinson Mary Ann
Published: June 2004

Measuring the diversity of TCRs utilized by specific primary and memory T cell responses is critical to the fundamental understanding of regulation of the immune system. This unit describes the Spectratype/Immunoscope technique which permits an in depth analysis of the TCR repertoire present in a variety of biological samples from mice to humans. Spectratype analysis takes advantage of PCR technology to amplify template cDNA corresponding to rearranged transcripts with different CDR3 lengths from specific TCR variable region genes in a competitive manner. The PCR products are then resolved on polyacrylamide sequencing gels to reveal precise sizes in nucleotide base pairs. The unit also includes protocols that have been optimized to process and produce the starting materials required for spectratype analysis.
Preparation and Analysis of Phosphorylated Proteins
Authors: Siegel Jeffrey N.
Published: June 2004

The function of cells of the immune system is regulated in part by engagement of specific receptors on the cell surface by soluble ligands or ligands presented on the surface of other cells. One of the major mechanisms by which cell‐surface receptor engagement influences cellular function is by activating protein kinases and increasing the phosphorylation of critical cellular proteins. There are two major categories of protein kinases: serine/threonine kinases which phosphorylate both serine and threonine residues; and tyrosine kinases which phosphorylate tyrosine residues exclusively. By metabolically labeling cells with 32P, as described in this unit, the phosphorylation state of cellular proteins can be analyzed and changes occurring with receptor stimulation can be examined. Phosphoamino acid analysis is also described to determine whether serine, threonine, or tyrosine residues are phosphorylated, thereby indicating which category of protein kinase is responsible. A protocol for phosphopeptide mapping is provided to allow the investigator to produce a “fingerprint” of the peptides that are phosphorylated, providing information regarding the sites of phosphorylation.
Assessment of the Effects of Tyrosine Protein Kinase Inhibitors
Authors: Burkhardt Anne L. , Bolen Joseph B.
Published: June 2004

The tyrosine protein kinases are enzymes that are important in cellular signal transduction. Therefore, inhibition of TPKs provides an important means of investigating and potentially controlling many signaling pathways. The first basic protocol in this unit describes an assay of the inhibitory effects of TPK inhibitors in vitro on a specific TPK that has been immune‐precipitated from cell lysates. An assay of the effects of several TPK inhibitors on TPKs in vivo in activated cells is also provided. Although the example used here is a nonreceptor TPK, these protocols can be used to assay the effects of inhibitors on receptor TPKs as well.
Isolation and Quantitation of HIV in Peripheral Blood
Authors: Koup Richard A. , Ho David D. , Poli Guido , Fauci Anthony S.
Published: June 2004

Quantitation of replication‐competent human immunodeficiency virus (HIV) in peripheral blood of infected individuals is critical for investigations of HIV pathogenesis and therapy. In this unit, the basic protocol determines the HIV titer in seropositive blood by measuring the tissue culture infectious dose (TCID) by an end‐point dilution method. A second basic protocol utilizes the PHA‐stimulated T cell blasts (activated T cells) in co‐culture with PBMC as described in the first basic protocol for the short‐term growth of HIV in vitro. An describes the accumulative method of determining 50% tissue culture infectious dose (TCID50) of HIV using the Reed‐Muench equation when multiple replicates of a given sample are employed in the assay. A consequence of HIV infection is the depletion of CD4+ target cells, evidenced by syncytia formation or single‐cell death; two support protocols detail the evaluation of these cytopathic effects.
Infection of CD4+ Primary Cell Lines and Induction of HIV
Authors: Poli Guido , Fauci Anthony S.
Published: June 2004

Chronically infected cell lines and clones are currently used as sources of virus to be used to infect other cell types as “factories” for large‐scale production of virus or viral components and as model systems for studies of the regulation of virus expression. In this unit, conditions for acute in vitro HIV infection are described. “Survivor” cells, resistant to the cytopathic effect of the virus, can be obtained from acute cultures. These survivor cells maintain an intact proliferative capacity and are considered to be chronically infected; their generation is described here. Finally, it is often desirable to study the effects of stimulants on HIV induction in these chronically infected cell lines; this procedure is detailed in a third protocol.
Regulation of the HIV Promoter/Enhancer
Authors: Palmieri Camillo , Trimboli Francesca , Scala Giuseppe , Quinto Ileana , Bressler Peter B.
Published: June 2004

This unit describes adaptations of two molecular techniques that can be used to study the regulation of HIV expression. The first two protocols describe the chloramphenicol acetyltransferase (CAT) assay, in which the CAT reporter gene is attached to an HIV‐1 promoter and CAT activity is measured as an indication of the promoter's activity. The basic protocol is rapid, simple, and suited to analyzing multiple samples. An alternate protocol describes an assay for CAT function that involves separating the reaction products by thin‐layer chromatography (TLC). The second basic protocol describes an electrophoretic mobility shift assay for detecting proteins present in cell extracts that can bind to the HIV‐1 LTR (long terminal repeat). Such studies are central to current HIV research because it is important to know what agents induce and inhibit (or “down‐regulate”) HIV transcription.
In Situ Hybridization for Detection of HIVRNA
Authors: Fox Cecil H. , Cottler‐Fox Michele
Published: June 2004

In HIV studies, in situ hybridization can be used for identifying virion RNA, mRNA being produced for virion packaging, and proviral DNA in the cytoplasm or integrated in the nucleus. This unit focuses primarily on identifying virion RNA, because this is the most sensitive means by which in situ hybridization can be employed to detect HIV expression. In situ hybridization, as developed for HIV RNA detection, involves several protocols: (1) preparation of a radioactive or nonradioactive RNA probe; (2) in situ hybridization of probe to cells and paraffin sections of tissue; (3) detection of radiolabeled probe by emulsion autoradiography; (4) development, staining, and mounting of slides; and finally (5) examination of slides by bright‐field, dark‐field, specular reflectance, or laser‐scanning confocal microscopy. The protocols presented in this unit describe a setup involving up to 150 slides.
In Vitro Evaluation of Experimental Agents for Anti‐HIV Activity
Authors: Richman Douglas D. , Johnson Victoria A. , Mayers Douglas M. , Shirasaka Takuma , O'Brien Mary C. , Mitsuya Hiroaki
Published: June 2004

This unit presents an assay that has proven useful as an initial screening test is an HIV cytopathic effect (CPE) inhibition assay in which immortalized T cell lines (e.g., ATH8 or MT2) that are profoundly sensitive to the cytopathic effect of certain strains of HIV are utilized as target cells. Additional protocols assess the anti‐HIV activity of certain candidate agents by measuring inhibition of syncytium formation or p24 gag protein production by ELISA. Calculation of the 50% inhibitory concentration (IC50) is also presented.
Vaccinia‐Based Reporter Gene Cell‐Fusion Assays to Quantitate Functional Interactions of HIV Envelope Glycoprotein with Receptors
Authors: Dey Barna , Berger Edward A.
Published: June 2004

This unit describes quantitation of functional interactions between HIV envelope glcoprotein and target cell receptors, using assay of cell fusion‐dependent reporter gene activation. The method is particularly useful since it isolates the fusion reaction from the rest of the HIV replication cycle, and obviates the need for infectious HIV particles.Reporter Gene Cell Fusion Assays to Quantitate Functional Interactions of HIV Envelope Glycoprotein with Receptors
Classical Pathway Evaluation
Authors: Giclas Patricia C.
Published: June 2004

This unit describes several assay methods that can be used to determine the functional status of the classical pathway of complement and to quantitate its component proteins. The classical pathway includes C1qrs, C2, C4, C3, C5, C6, C7, C8, and C9, listed in the order in which they interact. Two CH50 assay procedures are presented that test total classical pathway function; one is carried out in test tubes, whereas the alternate protocol describes a variation that is carried out in 96‐well microtiter plates. Three support protocols describe preparing serum and erythrocyte‐antibody complexes (EA) for use in CH50 assays,and titrating of hemolysin for use in EA preparation. As an alternative to functional assays, radial immunodiffusion (RID) methods are also presented. These can be used to measure the concentrations of most circulating complement proteins and to evaluate the functional status of C1‐esterase inhibitor. A fourth support protocol provides a method to determine antibody concentrations for RID assays.
Isolation and Purification of C3 from Human Plasma
Authors: O'Rear Lynda D. , Ross Gordon D.
Published: June 2004

The alternative pathway of complement shares its terminal components (C3 and C5 through 9) with the classical pathway, but has several unique components, including factors D, B, and P (properdin). This unit presents methods for assaying total alternative pathway activity and the activity of factors B and D. Radial immunodiffusion (RID) can also be used to measure factor D, B, and P concentrations.
Isolation of Murine Macrophages
Authors: Fortier Anne H. , Falk Lydia A.
Published: June 2004

Macrophages isolated from murine peritoneal cavity, bone marrow, and spleen are suitable samples for studying the activation properties of this immunologically important cell type. This unit describes the isolation of murine macrophages from the peritoneal cavity under inflammatory and noninflammatory conditions. To investigate macrophage activation using a more homogeneous population of cells, macrophages derived from immature progenitor cells in bone marrow can be studied. A second protocol describes the isolation of bone marrow‐derived progenitor cells and propagation of these immature macrophages by CSFs or IL‐3.
Characterization of Murine Macrophages
Authors: Riedy Mary C. , Stewart Carleton C.
Published: June 2004

This unit describes the phenotypic characterization of macrophages through the use of fluorescence‐labeled monoclonal antibodies (MAbs) that have specificity for certain extracellular membrane proteins expressed by macrophages. Although macrophages have no distinctive cell‐surface antigen that allows for their enrichment, a specific set of markers can be used to distinguish macrophages from other cell types in a heterogeneous population of cells. The method can also be easily applied to cells from other sources, e.g., peritoneal exudates or spleen. Through the use of image or flow cytometry, these cells can be identified and (with flow cytometry) sorted out from a heterogeneous population for further culture or analysis.
Characterization of Human Monocytes/Macrophages
Authors: Riedy Mary C. , Stewart Carleton C.
Published: June 2004

Human monocytes/macrophages display a variety of cell‐surface determinants to which monoclonal antibodies (MAbs) have been produced. These MAbs can be used to characterize phenotypic profiles of monocytes/macrophages to gain insight into their developmental and activation status. The basic and alternate protocols in this unit describe flow cytometry methods for measuring the expression of membrane determinants using directly conjugated or unconjugated MAbs, respectively. Because cells of this lineage display characteristic membrane marker patterns, combinations of MAbs are used in three‐color analysis to facilitate immunophenotypic characterization. Detailed information and commercial sources of fluorochrome‐labeled MAbs are provided, and suggested combinations of these MAbs for three‐color flow cytometry analysis of cell suspensions are discussed.
Activation of Murine Macrophages
Authors: Fortier Anne H.
Published: June 2004

Macrophages can act as potent effector cells of the immune response. Cytokine signals generated during an immune response activate macrophages in a complex cascade of events. Activated macrophages are qualitatively different from mature resident tissue macrophages and from immature inflammatory macrophages. Activated macrophages acquire new abilities to eliminate intracellular pathogens (bacterial or parasitic) and extracellular tumor or parasite targets. This unit describes the production of macrophages that display cytotoxicity against intracellular parasites, starting from resident peritoneal macrophages. An describes the generation of macrophages with tumoricidal activity, starting from inflammatory peritoneal macrophages.
Human Natural Killer Receptors and Their Ligands
Authors: Biassoni Roberto , Bottino Cristina , Cantoni Claudia , Moretta Alessandro
Published: June 2004

Human Natural Killer Receptors and Their Ligands (Roberto Biassoni and Cristina Bottino, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy; Claudia Cantoni, Universita degli Studi di Genova, Istituto Giannina Gaslini, Genova, Italy; Alessandro Moretta, Universita degli Studi di Genova, Genova, Italy). Natural killer (NK) cells are a lymphocyte subpopulation that are important effectors of innate immune responses against infectious pathogens. They are thought to play an important role in host defense, not only against virally infected cells, but also in killing of tumor cells. Recent progress indicates that NK cells express an array of receptors, some of them clonally distributed, able to modulate the natural cytotoxicity. Three NK‐specific activating receptors have been characterized; they belong to a novel receptor family called natural cytotoxicity receptors (NCR) and are represented by NKp46, NKp44, and NKp30. These receptors, upon engagement by their specific ligands, induce a strong activation of NK‐mediated cytotoxic activity. This overview discusses the receptors (both activating and inhibitory) expressed by NK cells and their ligands. Finally, the dysfunction of one of these molecules occurring in a genetically inherited immunodeficiency is discussed.
Isolation and Functional Use of Human NKT Cells
Authors: Exley Mark A. , Balk Steven P. , Wilson S. Brian
Published: June 2004

Isolation and Functional Use of Human NK T Cells (Mark A. Exley andSteven P. Balk, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts; S. Brian Wilson, Massachusetts General Hospital, Harvard Medical School, Cambridge, Massachusetts). This unit describes methods for the isolation and functional analysis of human invariant NK T cells. The first protocol uses standard multicolor flow cytometry methods and staining reagents specific for V to generate invariant NK T cell clones. Support protocols for secondary stimulations and for rapid expansion of invariant NK T cell clones and functional analysis of these cells are also included.
Collagen‐Induced Arthritis
Authors: Rosloniec Edward F. , Cremer Michael , Kang Andrew , Myers Linda K.
Published: June 2004

Collagen‐induced arthritis (CIA) is an experimental autoimmune disease that can be elicited in susceptible strains of rodents (rat and mouse) and nonhuman primates by immunization with type II collagen (CII), the major constituent protein of articular cartilage. Because of the important similarities between CIA and rheumatoid arthritis, this experimental model of autoimmune arthritis has been the subject of extensive investigation in several laboratories. Protocols for CIA are described in this unit for both the mouse model and the rat model. In addition, protocols are included for the purification of CII from bovine articular joints and chicken sternums, for the purification of collagen a1(II) chains, and for the purification of fragments of these chains following cyanogen bromide (CNBr) digestion. The preparation of CII is a time‐consuming procedure but is usually required because of the scarcity and expense of commercial sources of purified native CII. In addition, support protocols are provided for assessing the severity of inflammation following CIA and for measuring B and T cell responses to CII.
The NOD Mouse: A Model for Insulin‐Dependent Diabetes Mellitus
Authors: Leiter Edward H.
Published: June 2004

Nonobese diabetic (NOD) mice spontaneously develop autoimmune T cell‐mediated insulin‐dependent diabetes mellitus (IDDM). This unit presents a protocol for maintaining NOD mice under conditions permissive to full expression of their autoimmune potential. Methods are also described for diagnosing IDDM on the basis of glycosuria and glycosemia as well as for the semiquantitation of insulitis, a valuable subphenotype diagnostic of prediabetes in these mice, including a procedure for aldehyde fuchsin staining to identify β granules in β islet cells for diagnostic purposes. An adoptive‐transfer method is also included in which leukocytes, purified T cells, or T cell infiltrates obtained from the insulitic pancreas tissue of NOD mice are injected into prediabetic NOD or diabetes‐resistant F1 mice, which then develop disease in an accelerated fashion. This protocol also includes alternative steps in which bone‐marrow cells from NOD mice are transferred to syngeneic, irradiated NOD mice, allowing for reconsistution with a diabetogenic immune system. Steps for isolating pancreatic islet cells, which can then be used for a variety of purposes (e.g., as a source of islet antigens to establish and maintain autoreactive T cell lines) are included. Finally, steps are outlined that can be used to introduce transgenes into NOD mice. This protocol also discusses important considerations for introduction of targeted mutations produced in embryonic stem cells derived from other inbred strains, or introduction of other genes from non‐diabetes‐prone strains.
Induction of Autoimmune Disease by Depletion of Regulatory T Cells
Authors: Simmonds Steve , Mason Don
Published: June 2004

Organ‐specific autoimmune diseases can be induced in rodents that do not normally spontaneously develop autoimmunity by using procedures that render the animals partially T cell deficient. Using a protocol of adult thymectomy followed by four doses of sublethal gamma irradiation, insulin‐dependent diabetes can be induced in normal PVG.RT1u rats, an inbred congenic strain that has the same major histocompatibility complex (MHC) allotype as the spontaneously diabetic BB rat. Onset of the disease ranges from 3 to 18 weeks after the final dose of irradiation, with 98% of male and 70% of female animals becoming diabetic. This unit describes the induction of insulin‐dependent diabetes in the rat. A modified protocol allows for the induction of a more severe form of the disease.
Mercuric Chloride–Induced Autoimmunity
Authors: Badou A. , Saoudi A. , Dietrich G. , Druet E. , Druet P. , Pelletier L.
Published: June 2004

This unit describes methods for inducing autoimmune disease in Brown Norway rats through HgCl2 injections as well for assessing parameters that characterize the disease by serum IgE concentration assays, anti‐laminin antibody measurement, and renal immunofluorescence studies to detect autoantibodies. Also covered are disease induction using autoreactive CD4+ TH2 anti‐self MHC class II molecules and preparation of T cell lines. IL‐4 is produced very early after the first HgCl2 injection (beginning at day 3, peaking at day 14, and continuing up to day 30). Thus, IL‐4 mRNA expression may be detected in spleen and lymph nodes from HgCl2‐injected BN rats. The fact that HgCl2 induces in vitro mRNA IL‐4 gene expression in normal BN T cells but not in LEW T cells is probably crucial to susceptibility to the development of autoimmunity in the sense that it may condition the development of autoreactive T cells into pathogenic TH2 cells; a test for this condition is therefore also included.
The Day‐3 Thymectomy Model for Induction of Multiple Organ‐Specific Autoimmune Diseases
Authors: Suri‐Payer Elisabeth , Wei Kevin , Tung Kenneth
Published: June 2004

Autoimmune diseases of the ovary and the stomach are major components of the murine autoimmune polyendocrinopathy syndrome that occurs following experimental perturbation of the normal immune system. Several methods of perturbation can lead to autoimmune disease, and the disease occurs in multiple organs in association with autoantibodies to tissue‐specific antigen of the respective organs. The autoimmune diseases develop in a variety of laboratory mouse strains that are not prone to spontaneous autoimmune diseases. This unit describes the induction of autoimmune disease of the ovary and the stomach in mice by thymectomy at the age of day 3 (day‐3 thymectomy) and methods for evaluating disease histologically and serologically.
Animal Models for SLE
Authors: Cohen Philip L. , Maldonado Michael A.
Published: June 2004

Systemic lupus erythematosus (SLE) in humans is characterized by inflammatory lesions in skin, joints, kidneys, the central nervous system (CNS), and elsewhere. The clinical manifestations are accompanied by autoantibodies to diverse self antigens, mainly but not exclusively derived from the cell nucleus. Autoantibodies are generally believed to cause most of the tissue damage, although direct injury via cell‐mediated immunity is also important. This unit describes protocols for the quantitation of SLE‐associated autoantibody levels in mouse serum: detection of anti‐chromatin antibodies, detection of anti‐single and anti‐double‐stranded DNA antibodies, and detection of rheumatoid factor. Support protocols for preparing chicken chromatin and dsDNA are included. Also included is an immunoglobulin allotype‐specific adaptation of the basic autoantibody ELISA which is useful for measuring antibody production in chimeric animals. The unit includes an ELISPOT protocol for quantitating cells producing anti‐chromatin, as well as a method for genotyping mice for the faslpr and fasLgld mutations.