Flow cytometric analysis and sorting of human endometrial cells after immunocytochemical labeling for cytokeratin using a monoclonal antibody

Oud, Peter S.; Henderik, J. B. J.; Beck, H. L. M.; Veldhuizen, José A. M.; Vooijs, G. P.; Herman, Chester J.; Ramaekers, Frans C. S.

doi:10.1002/cyto.990060212

Cited by 46 publications

(16 citation statements)

References 13 publications

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“…The historical precedents for multiparam eter analysis of solid tumors came from stud ies of Braylan et al [3] which coupled analy sis of lymphoid cell surface antigens with DNA content measurements, and Darzynkiewicz et al [4] [1,5], endo metrium [6], and kidney [7], By coupling this approach with DNA analysis and gating on cytokeratin-positive cells, a more precise as sessment of proliferative activity and the presence of a rare DNA aneuploid popula tion among diploid neoplastic cells can be achieved. These cytoplasmic constituents, however, are lost for the most part in nuclear suspensions.…”

Section: Cellular Features Amenable To Flow Cytometric Analysis In Cementioning

confidence: 99%

Application of Multiparameter DNA Content and Immunofluorescence Analysis in the Investigation of Human Neoplasia

Bauer

1988

Path Immunopathol Res

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Section: Cellular Features Amenable To Flow Cytometric Analysis In Cementioning

confidence: 99%

Application of Multiparameter DNA Content and Immunofluorescence Analysis in the Investigation of Human Neoplasia

Bauer

1988

Path Immunopathol Res

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“…Recently, evidence has been found for extensive intratumor DNA index (DI) heterogeneity of breast carcinomas after multiple sampling (2,5). To improve the identification of carcinoma cells in heterogeneous tumor specimen by FCM, antikeratin antibodies have been used in combination with DNA stains (4, 26,28,32,39). This approach greatly improved the identification of tumor subpopulations and SPF determinations ( 30,(42)(43)(44)46,47).…”

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One-tube triple staining method for flow cytometric analysis of DNA ploidy and phenotypic heterogeneity of human solid tumors using single laser excitation

et al. 1996

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We have developed a “one‐tube” triple staining procedure that allows the identification of intratumor phenotypic subpopulations by FCM. Solid tumors were dissociated by a combined mechanical/enzymatic method. Ovarian ascites tumor cell aggregates were enzymatically dissociated using trypsin. An antikeratin 8/18 MAb was used to label the epithelial fraction of these tumor samples. A second MAb directed against the leukocyte common antigen (LCA) was applied to identify nonneoplastic DNA‐diploid cells. Other MAbs used as a second marker were directed against a tumor‐associated surface, a cytoplasmic, or a nuclear antigen. Cells were stained using subclass‐specific fluorescein‐isothiocyanate (FITC) or R‐phycoerythrin (PE)‐conjugated antibodies. DNA was stained with propidium iodide (PI). Triply stained samples were measured on a standard bench‐top flow cytometer (FACScan). Keratin 8/18‐positive cells, LCA‐positive cells, and DNA could be simultaneously detected in dissociated breast carcinomas, mixed Müllerian tumors, and ovarian ascites specimen for refining DNA index (DI) calculations and S phase fraction (SPF) determination. Coefficients of variation (CV) of the G0G1 peak of the DNA histograms obtained ranged from 2.55% to 4.64% and from 2.71% to 4.71% for the DNA‐diploid and ‐aneuploid fractions, respectively. In DNA‐diploid tumors, antigen expression (HER‐2/Neu, proliferating cell nuclear antigen) could be analyzed without interference of fluorescence signals from nonneoplastic cells. Neoplastic tumor subpopulations were clearly identified based on both DNA‐ploidy status and heterogeneity of antigen expression. The present method offers new possibilities for multiparameter DNA FCM on clinical samples and enables the identification of intratumor neoplastic subpopulations based on antigen expression and DNA‐ploidy status. © 1996 Wiley‐Liss, Inc.

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“…Occasionally, this paraformaldehyde fixation-induced effect is useful in identifying biologically distinct near-diploid subpopulations in tumors. 0 1992 Wiley-Liss, Inc.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.…”

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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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Flow cytometric analysis and sorting of human endometrial cells after immunocytochemical labeling for cytokeratin using a monoclonal antibody

Cited by 46 publications

References 13 publications

Application of Multiparameter DNA Content and Immunofluorescence Analysis in the Investigation of Human Neoplasia

Application of Multiparameter DNA Content and Immunofluorescence Analysis in the Investigation of Human Neoplasia

One-tube triple staining method for flow cytometric analysis of DNA ploidy and phenotypic heterogeneity of human solid tumors using single laser excitation

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

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