Detection of intracytoplasmic antigens by flow cytometry

Schroff, Robert W.; Bucana, Corazon D.; Klein, Richard; Farrell, Margaret M.; Morgan, Alton C.

doi:10.1016/0022-1759(84)90182-0

Cited by 67 publications

(37 citation statements)

References 12 publications

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“…Multiple samples, -150,000 cells each, were centrifuged onto glass slides, fixed in acetone, and then stained by utilizing the biotin-avidin-horseradish peroxidase technique as described above. Immunophenotyping of tissue sections and cytocentrifuge preparations after fixation reveals intracellular as well as surface antigen expression (25)(26)(27)(28) 10 Mg, and goat anti-mouse IgG (EY Labs, San Mateo, CA) at 4°C over 6 h while slowly rotating. Specific immunoprecipitation, using 10 ,g of specific, purified, anti-Leu-4 antibody and goat antimouse IgG at equivalence, was performed in a similar fashion over 18 h. Precipitates were collected by centrifugation and washed six times in NaCl-EDTA-Tris buffers containing NaCl 0.15 M, EDTA 5 mM, Tris-HCI 50 mM, pH 7.4, Na azide 0.02%, NP-40 decreasing from I to 0%, and additional NaCI to 0.5 M in the first four washes.…”

Section: Methodsmentioning

confidence: 99%

Discordance between surface and cytoplasmic expression of the Leu-4 (T3) antigen in thymocytes and in blast cells from childhood T lymphoblastic malignancies.

Link¹,

Stewart²,

Warnke³

et al. 1985

J. Clin. Invest.

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Section: Methodsmentioning

confidence: 99%

Discordance between surface and cytoplasmic expression of the Leu-4 (T3) antigen in thymocytes and in blast cells from childhood T lymphoblastic malignancies.

Link¹,

Stewart²,

Warnke³

et al. 1985

J. Clin. Invest.

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“…Schroff et al used lysolecithin in PBS as a permeabilizing agent for flow cytometric analysis of the p21 ras oncoprotein (22). We found extensive lysis of HL60 cells when they were permeabilized using the procedure of Schroff et al However, when lysolecithin is combined with 1% paraformaldehyde there is a significant improvement in cell morphology without loss of antibody accessibility to the cytoplasm or nucleus.…”

Section: Analysis Of Cell Surface and Pi-stained Cellsmentioning

confidence: 68%

“…The cells were fixed and permeabilized for 30 rnin at 4°C by incubating the cell pellets in 2 ml of various concentrations of lysolecithin (5 -40 pg/ml) dissolved in 1% paraformaldehyde. For comparison, HL60 leukemic cells were also permeabilized with lysolecithin (50 pg/ml) in PBS as described by Schroff et al (22). The cells were washed twice with PBS, pH 7.2 containing 1% BSA by centrifugation.…”

Section: Permeabilizationmentioning

confidence: 99%

Simultaneous paired analysis by flow cytometry of surface markers, cytoplasmic antigens, or oncogene expression with DNA content

Mc²,

Pryzwansky³

et al. 1989

Cytometry

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Flow cytometry is a useful tool for measuring DNA content and differentiation as expressed by cell surface markers. We have extended this technology to measure simultaneously either surface, cytoplasmic, or nuclear antigens (particularly oncoproteins) with DNA content. Mononuclear blood cells isolated from normal subjects and HL60 leukemic cells were permeabilized and fixed in suspension utilizing 40 pg/ml lysolecithin and 1% paraformaldehyde. A range of lysolecithin concentrations in 1% paraformaldehyde was studied to optimize permeabilization of the antibodies to the cell interior without destroying cell integrity. The optimal concentration (40 pg lysolecithidml) resulted in good cell recovery with a high percentage of cells positive for surface and intracellular antigens. Cells are first stained with fluorescein Flow cytometry is widely used as a method to quantify cell surface antigens and DNA content. It has less application for quantifying intracellular antigens because of the difficulty involved in introducing antibodies into cells without destroying antigenicity and cell integrity. Because of the heterogeneity of cell populations, techniques that enable one to correlate intracellular antigens with cell cycle kinetics could be useful. This is particdarly true for studying oncogene expression correlated with differentiation and cell cycle status.We report a method adapted for flow cytometry that correlates cell cycle kinetics with the amount of intracytoplasmic or intranuclear antigen. The technique utilizes lysolecithin and paraformaldehyde to permeabilize and fix cells simultaneously. With this technique cellular morphology is retained, allowing for the additional correlation of cell surface markers with DNA content. Leukemic cells (HL60 promyelocytic leukemia) were simultaneously stained to correlate DNA isothiocyanate conjugated (FITC) antimyeloperoxidase (an azurophil granule enzyme), or with an anti-c-myc antibody and FITC goat anti-mouse IgG F(ab')2. Cells are then incubated with RNase and stained for DNA content with propidium iodide. Alternatively, cells were stained for the cell surface markers Leu M3, OKM1, or the transferrin receptor and were then fixed and permeabilized and stained with propidium iodide. Using this method, we correlated cytoplasmic, nuclear, or cell surface antigens with cell cycle kinetics. This technique should be useful for studies of cellular differentiation and proliferation.

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“…Schroff et al (14) used lysolecithin, a naturally occurring phospholipid, to permeabilize lymphoid cells. Cells were then reacted with FITCconjugated antibodies in indirect binding assays to detect intermediate filaments.…”

Section: Discussionmentioning

confidence: 99%

“…The isolation of monoclonal antibodies cytometric measurements (14). that recognise antigenic determinants exposed at the In this report, studies that optimise techniques for surface of blood cell subpopulations and their applica-analysing internal antigens of D. discoideurn slug cells tion in flow cytometry has hastened this understanding are described.…”

Section: Key Terms: Monoclonal Antibody Permeabilisation Digitoninmentioning

confidence: 99%

Detergent treatment of dictyostelium discoideum cells allows examination of internal cell type‐specific antigens by flow cytometry

Bernstein

Browne

et al. 1988

Cytometry

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Monoclonal antibodies are used extensively in flow cytometry to identify subpopulations of cells differing in surface antigens. Conventional studies on living cells do not allow analysis of internal antigens, because antibody molecules do not pass through an intact plasma membrane. It is important for developmental studies on Dictgostelium discoideum that not only surface but also internal antigens be analysed. Here techniques are reported that make possible such studies by permeabilising cells with mild detergent treatments using digitonin. Over the past ten years there has been a revolution in culture cells that it is possible to permeabilise them so the understanding of the differentiation of lymphocyte that antibodies have access to internal antigens for flow subpopulations. The isolation of monoclonal antibodies cytometric measurements (14). that recognise antigenic determinants exposed at the In this report, studies that optimise techniques for surface of blood cell subpopulations and their applica-analysing internal antigens of D. discoideurn slug cells tion in flow cytometry has hastened this understanding are described. With mild detergent treatments, it is pos-(15). We have developed techniques for disaggregating sible to analyse internal antigens of unfixed cells. The the cells of the cellular slime mold Dictyostelium discoz-techniques outlined here are simple and rapid. We use deum slug, which is one of the simplest multicellular digitonin to permeabilise unfixed cells and to demoneukaryotes, and analysing them by flow cytometry (16). strate the effectiveness of this technique using two preOur studies centre on the differentiation of this organ-spore-specific internal antigens. ism, to produce two predominant cell types, the prespore and prestalk cells (16). In this way, cell surface antigens MATERIALS AND METHODS that identify prespore (9) or prestalk (10) cells have been Materials discovered.Digitonin (80% pure) was from Calbiochem. DeterIn recent work antigens that are cell type-specific but gents Triton X-100 and Nonidet P-40 were from Sigma. not found at the cell surface have been discovered (Alex-Monoclonal antibodies MUD1 (71, MUD3 (171, and ander et al., submitted). While internal antigens can be MUD102 (Smith, unpublished) raised against D. discoistudied after sorting the cells on the basis of cell surface markers, it would be more convenient to be able to detect internal antigens, directly in mixed-cell popula-'This research was supported by a NH & MRC (Australia) p a n t to tions, in the Same way that cell surface antigens are KLW. RLB was on sabbatical leave from San Francisco State studied by flow cytometry. Since antibodies cannot pen-u~~~~~r e p r i n t requests to Keith Williams, School of Biological etrate intact flow cytometry precludes such studies. However, it has been shown in tissue 2109.

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Detection of intracytoplasmic antigens by flow cytometry

Cited by 67 publications

References 12 publications

Discordance between surface and cytoplasmic expression of the Leu-4 (T3) antigen in thymocytes and in blast cells from childhood T lymphoblastic malignancies.

Discordance between surface and cytoplasmic expression of the Leu-4 (T3) antigen in thymocytes and in blast cells from childhood T lymphoblastic malignancies.

Simultaneous paired analysis by flow cytometry of surface markers, cytoplasmic antigens, or oncogene expression with DNA content

Detergent treatment of dictyostelium discoideum cells allows examination of internal cell type‐specific antigens by flow cytometry

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