bluuebunny/crossref_metadata_2025_split huggingface dataset:
A single laser method for subtraction of cell autofluorescence in flow cytometry
Authors: Alberti Saverio , Parks David R. , Herzenberg Leonard A. 
Published: August 2005

AbstractIn flow cytometry cell autofluorescence often interferes with efforts to measure low levels of bound fluorescent antibody. We have developed a way to correct for autofluorescence on a cell‐by‐cell basis. This results in improved estimates of real staining and better separation of the fluorescence histograms of stained and non‐stained cells.Using a single laser, two‐color fluorescence measurement system and two‐color compensation electronics, autofluorcscence and one fluorescent reagent are measured (rather than two fluorescent reagents). With fluorescein‐conjugated antibodies the signal in the 515 to 555 nm range (green fluorescence) includes both fluorescein emission and part of the cellular autofluorescence. In the cases we have investigated, autofluorescence collected at wavelengths above 580 nm (“red”) is well correlated with the green autofluorescence of the cells. A fraction of this red fluorescence is subtracted from the green fluorescence to produce an adjusted fluorescein output on which unstained cells have zero average signal.Use of this method facilitates the selection of rare cells transfected with surface antigen genes. Culture conditions affect the level of autofluorescence and the balance between red and green autofluorescence and the balance between red and green autofluorescence. When applied with fluorescein‐conjugated reagents, the technique is compatible with the use of propidium iodide for live/dead cell discrimination.
Trout and salmon erythrocytes and human leukocytes as internal standards for ploidy control in flow cytometry
Authors: Iversen O. E. , Laerum O. D. 
Published: August 2005

AbstractControl of technique and use of biological standards in flow cytometry have become increasingly important due to the wider use of the method for ploidy determination of malignant tumors in clinical research. Trout (TRBC) and salmon erythrocytes and human buffy coat leukocytes were selected for a study of factors influencing the DNA stainability. Whether standard and test cells were mixed before or after enzymatic treatment and staining was found to be critical for the ploidy comparisons. Otherwise, artifactual differences of at least 20% may be noted, leading to an overestimation of DNA aneuploidy. The time from staining to analysis had minimal effect, with some exceptions. The proportions of different cells in the sample had no influence, and nonlinearity of measurements was negligible. Diploid cells in normal endometrium and benign ovarian tumors, as well as the diploid fraction of aneuploid tumor cells, were systematically measured to have a DNA staining 5–7% above human leukocytes.
Cytophotometric determination of unscheduled DNA synthesis
Authors: Bose Kallol K. , Allison David C. 
Published: August 2005

AbstractWe describe a cytophotometric assay for unscheduled DNA synthesis (UDS) in asynchronously growing cells. Monolayer cultures of human HEp–2 and mouse MCa–11 cells were incubated with the carcinogen methylmethane sulfonate (MMS), as well as with hydroxyurea and (3H)thymidine. Slides were prepared, and the DNA contents and areas of nuclei were measured by absorption cytophotometry. The labeling of the nuclei, determined on the basis of their DNA content to be in G0/G1, was selectively measured after the preparation of autoradiographs. The labeling of the G0/G1 cells increased with increasing doses of MMS. We also found that the increased nuclear labeling after MMS treatment was not due to induction of replicative DNA synthesis or selective destruction of G0/G1 cells. The results of this assay compared favorably with a standard biochemical method for measuring unscheduled DNA synthesis by benzoylated naphthoylated DEAE cellulose chromatography.
An improved immunocytochemical procedure for high‐sensitivity detection of incorporated bromodeoxyuridine
Authors: Beisker Wolfgang , Dolbeare Frank , Gray Joe W. 
Published: August 2005

AbstractThis report describes an improved immunochemical procedure to stain cells in suspension for incorporated bromodeoxyuridine (BrdUrd) and total DNA content. The procedure consists of five steps: (1) chromatin proteins are extracted by treating with 0.1 M HCl and 0.7% Triton X‐100 to facilitate DNA denaturation and to minimize nonspecific staining; (2) cellular DNA is denatured by heating to 100°C in distilled water; (3) BrdUrd in single‐stranded DNA (ssDNA) is stained using an immunochemical procedure; (4) autofluorescence is reduced using sodium borohydride (NaBH4); and (5) DNA is stained with the fluorescent dye propidium iodide. With this procedure, the BrdUrd incorporated by CHO cells during periods as short as a few seconds can be detected using flow cytometry. In addition, the stoichiometry of the immunofluorescent staining procedure is high.
DNP‐phycobiliproteins, fluorescent antigens to study dynamic properties of antigen‐IgE‐receptor complexes on RBL‐2H3 rat mast cells
Authors: Seagrave Jean Clare , Deanin Grace G. , Martin John C. , Davis Bruce H. , Oliver Janet M. 
Published: August 2005

AbstractIn RBL‐2H3 rat mucosal mast cells, the crosslinking of cell‐surface IgE‐receptor complexes by multivalent antigens initiates a sequence of responses leading to degranulation. We have developed a family of dinitrophenol (DNP)‐conjugated fluorescent antigens to study dynamic membrane events associated with these responses. Lysyl groups on the phycobiliproteins, B‐phycoerythrin and C‐phycocyanin, were labelled with DNP, yielding fluorescent conjugates that cause the release of [3H]serotonin from anti‐DNO‐IgE‐primed RBL‐2H3 cells. The binding of these antigens to IgE‐receptor complexes was observed by fluorescence microscopy and quantified by flow cytometry. Incubation with 1 μg/ml DNP42‐B‐phycoerythrin stimulates maximum degranulation from IgE‐saturated cells. Under these conditions, approximately 26 × 103 molecules of DNP42‐B‐phycoerythrin are bound per cell at equilibrium. The rate and extent of antigen binding and of antigenstimulated mediator release decrease in parallel as the concentration and DNP:protein ratio of the fluorescent conjugates is reduced. Secretion stops immediately when the nonfluorescent monovalent antigen, DNP‐lysine, is added to degranulating cell suspensions. DNP‐lysine also displaces surface‐bound antigen when added during the first minutes after multivalent antigen. However, the ability of DNP‐lysine to displace surface‐bound DNP42‐B‐phycoerythrin from IgE‐receptor complexes decreases progressively with time. Treatment with dihydrocytochalasin B and several analogs that prevent antigen‐stimulated F‐actin assembly enhances secretion and delays the transition of antigen to its DNP‐lysine‐resistant form. Cytochalasin treatment also permits the long‐range movement of antigen into surface caps. Based on these data, we propose that secretion is triggered by the act of IgE‐receptor crosslinking or by a shortlived excited state of the crosslinked antigen‐IgE‐receptor complex. We propose further that antigen‐stimulated F‐actin assembly contributes to the transition of antigen‐IgE‐receptor complexes to a DNP‐lysine‐resistant form that does not trigger secretion. Two posible mechanisms for the transition to DNP‐lysine resistance are discussed.
Patterns of anthracycline retention modulation in human tumor cells
Authors: Krishan Awtar , Sridhar Kasi S. , Davila Enrique , Vogel Charles , Sternheim William 
Published: August 2005

AbstractLaser excitation of cellular doxorubicin and daunomycin content (with or without incubation in the presence of efflux blockers such as phenothiazines or verapamil) was studied in cells from leukemic peripheral blood, bone marrow aspirates, and ascites and pleural fluid of solid tumor patients. Selected examples are presented to show that heterogeneity in cellular anthracycline retention as well as sensitivity to efflux modulations is seen in human tumor cells. Several tumor subpopulations differing in their cellular retention of anthracyclines or sensitivity to modulators were seen. In serial tumor samples from patients (pre‐ and post‐treatment with anthra‐cycline‐containing protocols), initial drug retention andsensitivity of efflux modulators was followed by lack of drug retention and insensitivity to modulators. The present study shows that in view of the variables encountered in drug‐retention characteristics and sensitivity to efflux modulators, one needs to screen tumor samples before recommending use of any particular transport modulator to enhance drug retention and sensitivity of drug‐resistant cells.
Quantification of epithelial volume by image processing applied to ovarian tumors
Authors: Schipper N. W. , Smeulders A. W. M. , Baak J. P. A. 
Published: August 2005

AbstractThis paper describes an image analysis technique for automated estimation of the percentages of epithelium and stroma in (tumor) tissue. The program is evaluated on ovarian tumors of the serous, mucinous, and endometrioid type. From standard paraffin sections, stained with pararosanilin Feulgen and naphthol yellow, a blue‐yellow image pair was recorded. The blue image was used for the determination of the total tissue area and the yellow image for the epithelial area. For the latter the image processing method is based on the fact that epithelial nuclei are generally more tightly packed than stromal nuclei. A structural approach was applied, in which the segmentation of the nuclei was based on the image contrast range in thedensity domain. The method has been tested with 78 image pairs from 19 ovarian tumors with varying degrees of malignancy. The area percentages, as assessed with image processing, were strongly correlated to control percentages, established by interactive morphometry (r = 0.98).
A modular detector for flow cytometric multicolor fluorescence measurements
Authors: Steinkamp John A. , Habbersett Robert C. , Stewart Carleton C. 
Published: August 2005

AbstractA modular detector for measuring multicolor fluorescence from cells illuminated by single or multiple lasers has been developed for flow cytometers. Motion picture projector, camera, and CCTV/video lenses were evaluated for use in the detector by comparing their physical characteristics, image quality, and light collection efficiencies. A 25‐mm focal length F/0.95 CCTV lens was selected, based on our critera and test results. The detector was constructed out of square aluminum extrusion channels. A CCTV lens mounted on the outside of the first channel collected light emitted from cells and collimated it towards filters and secondary CCTV lenses located in each channel. The secondary lenses functioned as relay optics for directing and focusing light onto pinhole spatial filters for measurement by photomultipliers. The detector design allowed any number of channels to be connected together and the versatility for making simultaneous or sequential measurements. Measurements on lymphocytes labeled with four monoclonal antibodies conjugated to fluorescent dyes and measurements on viable tumor cells stained for DNA content and with three fluorescent‐labeled antibodies were used to demonstrate the detector's capabilities.
Use of tetrameric antibody complexes to stain cells for flow cytometry
Authors: Wognum Albertus W. , Thomas Terry E. , Lansdorp Peter M. 
Published: August 2005

AbstractBifunctional tetrameric complexes of monoclonal antibodies were used to stain cells for flow cytometry. These complexes consist of two different mouse monoclonal IgG1 antibodies (one with specificity for a cell surface antigen, the other with specificity for a fluorochrome) cross‐linked by two molecules of a monoclonal rat anti‐mouse IgG1. The use of this immunological approach to cross‐link fluorochromes to cell surface antigens was studied with tetrameric complexes containing Leu‐3a or Leu‐2a antibodies and monoclonal antibodies specific for the fluorochromes B‐ and R‐phycoerythrin. The ability of such cyclic immune complexes to stain T‐cell subset antigens on human peripheral blood lymphocytes was demonstrated in single and double‐staining experiments. The results demonstrate that tetrameric antibody complexes provide a simple and efficient alternative to covalently labeled antibodies for the flow cytofluorimetric analysis of cell‐surface antigens.
An improved method for the immunocytochemical detection of bromodeoxyuridine labeled nuclei using flow cytometry
Authors: Schutte B. , Reynders M. M. J. , van Assche C. L. M. V. J. , Hupperets P. S. G. J. , Bosman F. T. , Blijham G. H. 
Published: August 2005

AbstractA new staining protocol is described for the immunocytochemical detection of BrdUrd labeled nuclei. Pepsin treatment of ethanol fixed cells or tissue, followed by DNA denaturation at low pH, resulted in (1) increased sensitivity of BrdUrd staining comparable to the thermal denaturation protocol, and (2) decreased background binding. This technique is applicable to cell suspensions, including cultured cells and bone marrow cells. Furthermore, pepsin digestion of ethanol fixed tissue fragments resulted in a high recovery of nuclei in which incorporated BrdUrd could be detected. This possibility, together with the high sensitivity, make this method especially suitable for cell kinetic studies of human solid tumors in vivo.
Flow cytometric analysis of murine splenic B lymphocyte cytosolic free calcium response to anti‐IgM and anti‐IgD
Authors: Chused Thomas M. , Wilson H. Alexander , Greenblatt David , Ishida Yasuo , Edison Linette J. , Tsien Roger Y. , Finkelman Fred D. 
Published: August 2005

AbstractThe accurate measurement of ionized intracellular calcium [Ca++]i in single cells by flow cytometry with the use of a new fluorescent calcium chelator, indo‐1, is described. We have developed a dependable in situ calibration technique that indicates a resting [Ca++]i in lymphocytes of 100 nM. The enhanced fluorescence of this probe permits its use at sufficiently low cytoplasmic concentrations that buffering of [Ca++]i transients does not occur.The [Ca++]i response of small resting B lymphocytes to crosslinking of surface antigen receptors by anti‐immunoglobulin is heterogeneous. With maximal stimulus, the peak [Ca++]i response occurs in 10 to 20 seconds with most cells reaching levels >/1 μM. Mean [Ca++]i falls to between 300 and 800 nM by 100 seconds where it remains for more than 10 min. Anti‐δ is a more potent stimulus of increased [Ca++]i than anti‐μ in terms of both [Ca++]i level and fraction of B cells responding. Whether this is due to the greater density of surface IgD than IgM, a difference in signal transduction efficiency, or both, is not yet known. Surface immunoglobulin receptors are present in great excess. Less than 3% of surface immunoglobulin is crosslinked at the peak of the [Ca++]i response.
Anomalous behaviour of forward and perpendicular light scattering of a cyanobacterium owing to intracellular gas vacuoles
Authors: Dubelaar George B. J. , Visser Jan W. M. , Donze Marcel 
Published: August 2005

AbstractExtinction, absorption, and forward and perpendicular light scatter of the blue‐green alga Microcystis aeruginosa with different amounts of intracellular gas vacuoles were determined. The amount of gas vacuoles in the cells was controlled by application of pressure. The presence of the gas vacuoles caused a tenfold increase in perpendicular light scatter, and a fivefold decrease in forward light scatter as measured by flow cytometry. Chlorophyll fluorescence showed a 16% decrease. The presence of gas vacuoles did not affect the size of the algae. The absorption spectrum of Microcystis aeruginosa was slightly raised but practically not distorted by the gas vacuoles. The attenuation spectrum, a measure for light extinction by the algal cells, was significantly distorted. The increase of perpendicular light scatter intensity of the cells is a direct consequence of the relatively high scatter of each vacuole, whereas the forward light scatter decrease is attributed to a lower phase‐shift factor s̀ of the whole cells, caused by the intact gas vacuoles.
Discrimination of viable and non‐viable cells using propidium iodide in two color immunofluorescence
Authors: Sasaki Dennis T. , Dumas Stephanie E. , Engleman Edgar G. 
Published: August 2005

AbstractThe relative ease with which a flow cytometer can perform simultaneous two color immunofluorescence to examine subpopulations of lymphoid cells has been well documented. Thus, flow cytometers equipped with only a single argon laser can be used to delineate various cell types by exciting both fluorescein‐ and phycoerythrin‐conjugated antibodies to cell surface antigens. One problem that remains, however, is the artifactual staining of dead cells and clumps, which cannot be distinguished from viable cells on the basis of cell surface staining characteristics. We describe a method for simultaneous two color analysis or sorting of viable leukocytes which requires only a single laser. The method utilizes propidium iodide, which stains dead cells and thereby excludes such cells from the analysis. Using this method, as many as four viable cell types have been simultaneously analyzed in a single sample.
Viable sorting of intact multicellular spheroids by flow cytometry
Authors: Freyer James P. , Wilder Mark E. , Jett James H. 
Published: August 2005

AbstractA flow cytometric method has been developed for sorting viable, intact multicellular spheroids in order to obtain uniformly‐sized populations with diameters in the range of 50–100 μm. A FACS II instrument was modified for this purpose by installing a 200‐μm‐diameter exit orifice and by making adjustments in the sheath flow, oscillator frequency, and number of droplets sorted. Polystyrene microspheres (44 and 88 μm diameter) and 41–96‐μm‐diameter spheroids could be sorted and recovered with 70–100% efficiency, an improvement over previous reports. Unstained, viable spheroids were simultaneously analyzed for small‐angle forward light scatter, 90° light scatter, and autofluorescence using a 488‐nm laser operating at 100 mW. Analysis of the data demonstrated a considerable variation in both the 90° light scatter and the autofluorescence signals for a given forward angle light scattering signal. By setting narrow sort windows on the forward angle light scattering signal and either the 90° light scatter or autofluorescence signals, uniformly spherical spheroid populations could be recovered. These sorted populations had coefficients of variation of the mean diameter in the range of 5–9%. This represents a variation of less than one cell diameter, and is a major improvement over any other technique. There was no significant difference in the subsequent growth rates of sorted spheroids compared to the unsorted spheroids. This technique will apply when uniform populations of small spheroids are required, such as in investigations of the contact effect or in the initiation of growth curve studies.
A comparison of low‐power helium‐cadmium and argon ultraviolet lasers in commercial flow cytometers
Authors: Bigler Robert D. 
Published: August 2005

AbstractThe feasibility of installing a low power ultraviolet (UV) laser in a commercial flow cytometer was evaluated by testing an Ortho Cytofluorograf 50HH and a Coulter Epics V. Both instruments were equipped with two argon ion lasers, one emitting at 488 nm and the other in the UV region and were tested by measuring the DNA content of cells stained with Hoechst 33342 or DAPI. The coefficient of variation (CV) of the G0/G1 peak of the DNA histograms produced by each instrument did not deteriorate markedly when results obtained at 100–125 mW were compared to those obtained at 10 mW. These tests indicated that a helium‐cadmium laser (He‐Cd) which can produce 10 mW at 325 nm should work well as a UV laser in these instruments. An Ortho Cytofluorograf IIs was purchased with a 10 mW He‐Cd laser installed in the forward position. Studies of DNA content have confirmed that this low power UV laser can produce CVs of 2.2% with DAPI stained fixed cells and 3.6% with Hoechst 33342 stained viable lymphocytes. Thus, the He‐Cd laser should provide a reasonable alternative as a UV source for flow cytometers.
Flow cytometric purification of Alzheimer's disease amyloid plaque core protein using thioflavin T
Authors: Palutke Margarita , Kukuruga Debra , Wolfe David , Roher Alex 
Published: August 2005

AbstractAmyloid plaque core protein (APCP) of Alzheimer's disease obtained from brain tissue homogenate is difficult to recover in pure form, primarily because of contaminating lipofuscin (LF) granules. Thioflavin T, a fluorescent dye previously used to stain amyloid, was found to bind to APCP but not to lipofuscin. The latter, however, is autofluorescent. Fluorometric studies showed that at 370 nm excitation APCP has a maximal emission at 418 nm, whereas the autofluorescent LP has a maximal emission at 450 nm. This difference in emission permitted the use of a flow cytometer‐sorter (FACS 440) for purification of APCP. APCP particles fluoresced distinctly from LF granules on the log blue fluorescence parameter. The two entities were sorted using forward light scatter versus fluorescence. A collection apparatus was designed and prepared to facilitate the collection of large volumes of sheath fluid and particles and to minimize fragmentation of particles during the collection process. The sorted APCP fraction was 98% pure. This work demonstrates how old dyes can be used to perform new tricks and provide a useful method for separating complex protien.
Immunofluorescent quantification of ribonucleotide reductase M1 subunit and correlation with DNA content by flow cytometry
Authors: Mann Graham J. , Dyne Marlen , Musgrove Elizabeth A. 
Published: August 2005

AbstractThe cytoplasmic enzyme ribonucleotide reductase is essential for DNA synthesis, and its activity is strongly correlated with cellular proliferation. This paper describes a flow cytometric technique for the simultaneous measurement of DNA content and the M1 subunit of ribonucleotide reductase. Data are presented for cycling cultured human leukemic lymphoblasts in which M1 is constitutively expressed, and peripheral blood lymphocytes in which it is only detectable with certainty after mitogen stimulation.The choice of fixation procedure strongly influenced the amount of M1 subunit detected. Paraformaldehyde (PF) at concentrations of 2% (w/v in PBS) or greater provided optimal results. Fixation at 37°C was significantly more effective in preserving M1 than fixation at room temperature or 4°C. These variables are shown to have affected cytoplasmic retention during postfixation processing. Their relevance to the flow cytometric measurement of other intracellular components by this procedure are discussed.
Spectral analysis of flow cytometric data: Design of a special‐purpose low‐pass digital filter
Authors: Sloot Peter M. A. , Tensen Pieter , Figdor Carl G. 
Published: August 2005

AbstractSpectral decomposition of flow cytometric datafiles of arbitrary dimension reveal information of both the signal and the noise components that constitute the histograms. This spectral information is used to construct a low‐pass digital filter, which removes the high‐frequency noise from the actual data. It is shown that this procedure guarantees nontrivial smoothing of the flow cytometric data in accordance with the local experimental situation. As a consequence optimal reconstruction of the signal is possible, which facilitates unambigous interpretation of the data files and mathematical estimation of the statistical parameters.