Identification of live cells for flow cytometric analysis of lymphoid subset proliferation in low viability populations

Muirhead, Katharine A.; Kloszewski, E D; Antell, Laura; Griswold, Don E.

doi:10.1016/0022-1759(85)90185-1

Cited by 18 publications

(12 citation statements)

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“…Two methods for measurement of cell-cycle distributions by DNA flow cytometry combined with dead cell exclusion have been described previously (4,37), but each has a disadvantage. The technique described by Muirhead et al requires preincubation of the cells before staining with deoxyribonuclease to permit discrimination of dead cells from live cells by decreased DNA content (4); however, this step adds approximately 90 min to the sample preparation time.…”

Section: Discussionmentioning

confidence: 99%

“…analysis by differential light scatter gating. When cells have been cultured in vitro, however, these differences are often not clear enough to permit accurate dead cell exclusion (4). A further loss of distinction in scatter parameters between viable and nonviable cells is usually observed in cell preparations that have been fixed and/or permeabilized (4,10,11).…”

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confidence: 99%

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Flow cytometric analysis of live cell proliferation and phenotype in populations with low viability

Schmid¹,

Ferbas

Uíttenbogaart

et al. 1999

Cytometry

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Background: Combined analysis of DNA content and immunofluorescence on single cells by flow cytometry provides information on the proliferative response of subpopulations to stimuli in mixed cell preparations; however, in low‐viability cell preparations, dead cells interfere with accurate flow cytometric data analysis because of nonspecific binding of antibodies and altered DNA‐staining profiles. Light scatter differences between nonviable and viable cells are unreliable, particularly after the cell permeabilization step that is necessary for DNA staining. We developed a method for identification of nonviable cells by fluorescence in cell preparations that are stained simultaneously for cell surface or intracellular immunofluorescence and DNA content. Materials and Methods: Nonviable cells that have lost membrane integrity are identified by uptake of 7‐amino‐actinomycin D (7‐AAD). Transfer of 7‐AAD from stained nonviable cells to unstained viable cells after permeabilization is prevented by blocking DNA binding with nonfluorescent actinomycin D (AD). Pyronin Y(G) (PY) is used for DNA staining because the orange spectral emission of PY can be separated from the green fluorescein isothiocyanate (FITC) emission and the red emission of 7‐AAD, respectively. Results: Application of the method to the analysis of the T‐cell leukemia cell line Molt‐4f and of cultured human peripheral blood mononuclear cells is presented. In both cell preparations, 7‐AAD staining permitted reliable dead cell exclusion. Live, 7‐AAD–negative Molt‐4f cells showed higher expression levels of cell surface CD4 and of intracellular CD3, showed a higher proportion of cells in the G1 phase of the cell cycle, and showed a lower coefficient of variation of the G1 peak compared with data obtained from all the cells in the preparation. Live, CD8+ lymphocytes from OKT3‐stimulated cultures of human peripheral blood mononuclear cells showed a specific proliferative response as measured by DNA content analysis. Conclusions: The results show that cells stained with FITC–labeled antibodies can be analyzed by single‐laser flow cytometry for DNA content combined with dead cell discrimination. Furthermore, they emphasize the need for exclusion of dead cells from the analysis of cell preparations with low viability to obtain reliable data on immunofluorescence and cell‐cycle distributions. Cytometry 35:64–74, 1999. © 1999 Wiley‐Liss, Inc.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Flow cytometric analysis of live cell proliferation and phenotype in populations with low viability

Schmid¹,

Ferbas

Uíttenbogaart

et al. 1999

Cytometry

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“…One reason for this is a concern that dead or dying cells may nonspecifically bind monoclonal antibodies used for immunophenotyping or compromise analysis due to autofluorescence (16, 17). In addition, discrimination against dead or dying cells by forward and light scatter properties, although in common use, may not be accurate (18, 19) and may lead to inclusion of variable proportions of such cells in the proliferation analysis. It is therefore preferable to identify apoptotic or dead cells with the use of specific dyes (20).…”

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Five‐color flow cytometric analysis of swine lymphocytes for detection of proliferation, apoptosis, viability, and phenotype

2002

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Background:The objective of this study was to develop a method to simultaneously examine phenotype, proliferation, apoptosis, and death of antigen-stimulated porcine lymphocytes. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from pigs vaccinated with a Brachyspira hyodysenteriae bacterin. Results: Once isolated, PBMCs were stained with the fluorescent membrane intercalating dye, PKH67, and cultured with or without B. hyodysenteriae whole-cell sonicate antigen. Serial samples of nonstimulated and B. hyodysenteriae-stimulated PBMCs were harvested for flow cytometric analysis. Fluorochrome excitation was performed with spatially separated air-cooled argon and red helium neon laser beams. Five-color analysis included signal detection of PKH67 (proliferation), phycoerythrin

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“…Historically, lymphoid and myeloid cell surface antigens have been quite sensitive to the effects of commonly used methods of chemical fixation (3,(5)(6)(7)(8)(9)(10)(11)(12). These were generally short-term studies.…”

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confidence: 99%

“…These were generally short-term studies. Chemical fixation after immunostaining has been found of value in prolonging the time frame in which cells may be subsequently analyzed by flow cytometry (3,(5)(6)(7)(8)(9)(10)(11)(12). However, it has thus far not been possible to fix cells for long-term storage and then undergo retrospective immunostaining and analysis.…”

mentioning

confidence: 99%

Preservation of B‐cell‐associated surface antigens by chemical fixation

Caldwell

1994

Cytometry

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Identification of live cells for flow cytometric analysis of lymphoid subset proliferation in low viability populations

Cited by 18 publications

References 6 publications

Flow cytometric analysis of live cell proliferation and phenotype in populations with low viability

Flow cytometric analysis of live cell proliferation and phenotype in populations with low viability

Five‐color flow cytometric analysis of swine lymphocytes for detection of proliferation, apoptosis, viability, and phenotype

Preservation of B‐cell‐associated surface antigens by chemical fixation

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