Analysis of intracellular antigens by flow cytometry

Jacobberger, James W.; Fogleman, David; Lehman, John M.

doi:10.1002/cyto.990070410

Cited by 117 publications

(66 citation statements)

References 32 publications

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“…The duration of exposure to paraformaldehyde was evaluated over a range of 15 min t o 2 h. Once cells were fixed in paraformaldehyde at lor above room temperature for at least 15 The effects of methanol concentration and the duration of cell exposure to methanol were not found to be of critical importance, provided that methanol concentration exceeded 22.5%, and the duration of exposure was 1 h or longer. We routinely expose cells to 70% methanol for 1 h a t 4°C.…”

Section: Optimal Cell Fixation Conditions For Immunofluorescence Studmentioning

confidence: 99%

Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins

et al. 1992

Cytometry

111

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A cell fixation and permeabilization procedure consisting of sequential paraformaldehyde and methanol was evaluated and found suitable for concomitant flow cytometric quantification of total cellular DNA, immunofluorescence measurements of cell surface proteins, and immunofluorescence measurements of intracellular proteins. Paraformaldehydeimethanol-fixed cells exhibited significantly greater intracellular antitubulin immunofluorescence than cells fixed with paraformaldehyde or methanol alone 0, < 0.002) and significantly greater intracellular antitubulin immunofluorescence than cells fixed with methanol followed by paraformaldehyde 0, < 0.006).With paraformaldehyde/methanol fixation, cell morphology was well preserved and forward and right angle light scatter properties were sufficiently well maintained to permit gating on these parameters. Cell surface marker staining with fluorescent anti-leukocyte antibodies was unaffected by fixation with paraformaldehyde/methanol. Paraformaldehyde effects on the intensity of DNA staining with propidium iodide were dependent on paraformaldehyde concentration and fixation temperature; these effects were least pronounced at low paraformaldehyde concentrations (0.25% or less), and at temperatures lower than 37°C. Paraformaldehyde fixation may result in differences in propidium iodide staining of DNA in some diploid cells, which may produce small spurious aneuploid peaks in normal peripheral blood leukocytes. Paraformaldehyde fixation also produces an apparent increase in the DNA index of aneuploid cell populations in comparison with methanol fixation, particularly when the DNA index exceeds 1.5. Occasionally, this paraformaldehyde fixation-induced effect is useful in identifying biologically distinct near-diploid subpopulations in tumors. 0 1992 Wiley-Liss, Inc.Key terms: Flow cytometry, cell fixation, membrane permeabilization, paraformaldehyde, methanol, multiparameter analysis, immunofluorescence There are many published studies in which specific intracellular proteins have been studied quantitatively by means of flow cytometric immunofluorescent techniques (1,(15)(16)(17)(18)(19)(20)(21)(22)(23)25), often in combination with cellular DNA content. Unfortunately, no clear consensus has emerged from these studies with regard to optimal cell fixation conditions for multiparameter flow cytometric analysis. In a number of studies, cells were fixed in methanol alone (8,15,19) or ethanol alone (1,3,7,9,10,16,18,25). However, comparative studies of alcohol with paraformaldehyde fixation have suggested that there may be significant loss of cytoplasmic or nuclear proteins from cells following fixation with alcohol alone (17,181.

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Section: Optimal Cell Fixation Conditions For Immunofluorescence Studmentioning

confidence: 99%

Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins

et al. 1992

Cytometry

111

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“…A detailed description has been published (13) of the staining procedure. Briefly, after removal of fixative, the cells were resuspended in anti-large T (PAB 101) diluted in PBS-NGS (PBS-145 mM NaC1, lOmM phosphate, pH 7.4, and equal volume NGS -heat inactivated normal goat serum with 0.002% Triton X 100 and 0.1% sodium azide).…”

Section: Fixation and Stainingmentioning

confidence: 99%

Analysis of simian virus 40 infection of CV‐1 cells by quantitative two‐color fluorescence with flow cytometry

et al. 1988

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Key terms: DNA content changes, permissive infection, viral antigens, T-antigen, V-antigen Considerable interest has been generated in the analysis of gene products obtained from both viral and cellular oncogenes, since these genes may be responsible for such activities as cell proliferation, differentiation, and the acquisition of the neoplastic phenotype (1). Simian Virus 40 (SV40) has been an important model system for studies of viral replication, viral transformation, and gene regulation (14,20,22). The early viral gene products large T and small t antigen are thought to be responsible for events in viral replication in permissive cells and also for the initiation and establishment of the neoplastic phenotype in susceptible nonpermissive cells (4). The SV40 T antigen is known to bind viral DNA at the origin of replication and initiate viral DNA synthesis in the permissive cell (19,26). Further, the T antigen activates late viral transcription and inhibits early viral transcription (2). The part that the T antigen plays in transformation may be at a number of levels which include gene regulation and the interactions with the plasma membrane and cellular proteins such as p53 (15,25,27). The molecular approach to the study of viral expression has provided much information concerning the role of these proteins in permissive and nonpermissive cells.One further method of investigation would utilize flow cytometry to quantitatively assay the appearance of nity to assay various parameters of a population of cells, such as DNA and specific viral and cellular proteins, and to correlate these parameters on a single-cell basis (3,5,17,29). The instrumentation also allows quantitation of each of these parameters singly and in concert. In previous studies, a technique was developed that allowed simultaneous staining of cellular DNA content and the viral T antigen using a specific monoclonal antibody (13). This technique has been applied to study SV40 infection of permissive CV-1 cells. Changes in host cell cycle distribution and the synthesis of both T antigen and the late viral proteins that encapsulate the viral DNA were followed in time. MATERIALS AND METHODSCells and Virus The cells utilized in this study were the CV-1 line of African green monkey kidney, passages 26 through 38, CV-1 cells were grown in modified Eagle's medium (MEM, GIBCO) with 2 x amino acids and vitamins. MEM with 10% fetal bovine serum (FBS) was used for growth and 5% FBS for maintenance. The CHE lines were grown in MEM with 5% FBS. Cultures were kept at 37°C in humidified 5% C 0 2 incubator (17).The SV40 stock virus used was RH911, grown and plaque-assayed for titer on CV-1 cells. All cells and virus stock tested negative for mycoplasma contamination (17).Infection was performed on 100-mm plastic petri dishes containing CV-1 cells that were maintained for 1-2 days after reaching confluence. At this time, media was removed and 1.0 ml of virus, diluted in MEM, was added to the cultures. The cells were infected with varying multiplicities of infecti...

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“…An increasing number of papers have described flow cytometric analyses of intranuclear antigens (reviewed 0 1987 Alan R. Liss, Inc. in reference 8) and several have presented simultaneously acquired DNA histograms of high quality (2,8,17). However, reports concerning cytoplasmic antigens are relatively rare and none have explicitly sought to optimize both immunofluorescence and DNA staining (1,7,11,13,14,18,19).…”

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Immunofluorescent quantification of ribonucleotide reductase M1 subunit and correlation with DNA content by flow cytometry

Mann¹,

Dyne²,

Musgrove³

1987

Cytometry

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The 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% (wlv in PBS) or greater provided optimal results. Fixation at 3'7°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.Key terms: Cell cycle, cytoplasmic proteins, paraformaldehyde, propidium iodide Ribonucleotide reductase catalyzes the conversion of ribonucleoside diphosphates to their corresponding deoxyribonucleotides. This is the first unique, rate-limiting step in DNA synthesis. Biochemical studies have shown that the activity of this enzyme is high in proliferating tissues and suggest that catalysis only takes place during S phase (16).Recently, monoclonal antibodies have been raised against the ribonucleotide reductase M1 subunit (a polypeptide of Mr 80,000 which binds allosteric effectors) and have been used in a sensitive biochemical immunoassay and in histochemical studies (3,5,6). The latter work showed that the M1 subunit is cytoplasmic and detectable only in proliferating tissues. We therefore sought to develop a flow cytometric technique to quantify ribonucleotide reductase M1 subunit levels by immunofluorescence, correlate these measurements simultaneously with DNA content, and directly examine cell cycle effects on enzyme content.Flow cytometric techniques for surface immunofluorescence studies are well established, but measurement of intracellular antigens is less easy to achieve because of the need to render the cell permeable to antibody without denaturing or dispersing the target molecule. An increasing number of papers have described flow cytometric analyses of intranuclear antigens (reviewed 0 1987 Alan R. Liss, Inc. in reference 8) and several have presented simultaneously acquired DNA histograms of high quality (2,8,17). However, reports concerning cytoplasmic antigens are relatively rare and none have explicitly sought to optimize both immunofluorescence and DNA staining (1,7,11,13,14,18,19).We here report a n optimized procedure for demonstrating the cytoplasmic protein, ribonucleotide reductase M1 subunit, by flow cytometry. It is based on formaldehyde fixation because this antigen is poorly preserved by other cytological fixatives in this context. Though aldehydes can involve a compromise of DNA staining...

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Analysis of intracellular antigens by flow cytometry

Cited by 117 publications

References 32 publications

Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins

Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins

Analysis of simian virus 40 infection of CV‐1 cells by quantitative two‐color fluorescence with flow cytometry

Immunofluorescent quantification of ribonucleotide reductase M1 subunit and correlation with DNA content by flow cytometry

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