A simplified method for the coordinate examination of apoptosis and surface phenotype of murine lymphocytes

Douglas, Raymond S.; Tarshis, Adam D.; Pletcher, Charles H.; Nowell̀, Peter C.; Moore, Jonni S.

doi:10.1016/0022-1759(95)00216-2

Cited by 38 publications

(31 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It requires only a small number of cells, fluorescent probes are now available for the analysis of many aspects of cell proliferation, differentiation, and activation, and it also permits the simultaneous investigation of cells of different phenotypes within the same sample. Among the more common methods for measuring apoptosis by flow cytometry are those based on cell size and granularity and on DNA content analysis (8,13,24,25).…”

Section: Discussionmentioning

confidence: 99%

“…The appearance of this peak is a specific marker of apoptosis: necrosis induced by metabolic poisons or lysis induced by complement did not induce any sub-G1 peak in the DNA. The sub-G1 peak method is simple, quantitative, and applicable to all cell types, but has a main limitation: it is not adequate for simultaneous immunophenotyping due to the ethanol-related alterations of the lipid membrane and protein structure (24).…”

Section: Discussionmentioning

confidence: 99%

“…Although several methods have been developed for the simultaneous examination of apoptosis and surface phenotype of lymphocytes (24,26,27), the main advantage of our method is the use of unfractionated whole blood as the source of cells for testing susceptibility to apoptosis. Usually, the first step in performing a cytometric evaluation of apoptosis is the separation of PBMC through a density gradient (22).…”

Section: Discussionmentioning

confidence: 99%

“…One study using human mature T cells reported that freshly isolated, by contrast to short-term cultured, T cells were not sensitive to glucocorticoid-induced apoptosis (28). On the other hand, apoptosis induced by glucocorticoids in vitro in freshly isolated human (13) and mouse (12,24) lymphocytes has been reported. The higher sensitivity for glucocorticoid-induced apoptosis of CD8ϩ T cells in relation to CD4ϩ T cells observed in our study has been described previously in the context of isolated human T cells (28).…”

Section: Discussionmentioning

confidence: 99%

See 3 more Smart Citations

A small‐volume technique for simultaneous immunophenotyping and apoptosis detection in rat whole blood by four‐color flow cytometry

2002

View full text Add to dashboard Cite

BackgroundCell permeabilization for the detection of intracellular molecules by flow cytometry is usually incompatible with whole blood. This article describes a new technique for the simultaneous detection of surface antigens and DNA content in rat whole blood.MethodsIn 20 μl of rat whole blood, DNA staining is obtained by permeabilization of cells using a standard red blood cell lysing reagent (Erythrolyse). Immunophenotyping and apoptosis detection by flow cytometry are achieved by using a combination of three surface markers (CD3, CD4, and CD8α) and a DNA binding dye (TO‐PRO‐3).ResultsAfter a 24‐h incubation of whole blood with 1 μM dexamethasone, apoptotic lymphocytes were clearly distinguishable from normal lymphocytes by their reduced size and DNA content. The dexamethasone‐induced percentage of apoptotic cells was 58.9 ± 4.6 for CD4+ and 77.4 ± 2.9 for CD8+ T cells, compared with 12.6 ± 2.7 for CD4+ and 17.2 ± 3.5 for CD8+ T cells in the absence of dexamethasone (data from 10 animals with duplicate samples).ConclusionsWe have developed a new technique to permeabilize nucleated cells in microsamples of rat whole blood. The methodology allows simultaneous immunophenotyping and apoptosis detection in rat whole blood. Cytometry 47:265–275, 2002. © 2002 Wiley‐Liss, Inc.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

A small‐volume technique for simultaneous immunophenotyping and apoptosis detection in rat whole blood by four‐color flow cytometry

2002

View full text Add to dashboard Cite

show abstract

“…Flow cytometric analysis of IRF4-positive cells and cell cycle The intracellular expression of IRF4 protein was analyzed by flow cytometry in fixed and permeabilized cells. 25) Briefly, 3-5×10 5 cells were fixed with ice-cold 1% paraformaldehyde for 4 min, permeabilized with 0.2% Tween 20 at 37°C for 15 min, and then washed twice with phosphate-buffered saline (PBS) containing 2% FBS (FBS/PBS). Cells were incubated at 4°C with fluorescein isothiocyanate (FITC)-conjugated anti-human IRF4 MAb or isotype control Ab (PharMingen, San Diego, CA) for 60 min, washed and suspended in FBS/PBS, and analyzed by FACScan using the CELLQuest software (Becton Dickinson, San Jose, CA).…”

Section: Methodsmentioning

confidence: 99%

Possible Involvement of Interferon Regulatory Factor 4 (IRF4) in a Clinical Subtype of Adult T‐Cell Leukemia

Imaizumi

Kohno

Yamada

et al. 2001

Japanese Journal of Cancer Research

View full text Add to dashboard Cite

Interferon regulatory factor (IRF) 4 is the lymphoid-specific transcription factor that is required for the proliferation of mitogen-activated T cells. IRF4 has been suggested to be involved in tumorigenesis because the overexpression of IRF4 caused the transformation of Rat-1 fibroblasts in vitro. Here, we show that IRF4 is constitutively expressed in adult T-cell leukemia (ATL)-derived cell lines, which were infected with human T-cell leukemia virus type-I, but hardly expressed the trans-activator protein, Tax. Similarly, constitutive expression of IRF4 was demonstrated in freshly isolated peripheral blood mononuclear cells (PBMC) from patients with either acute or chronic ATL. However, the high-level expression of IRF4 was specifically associated with acute ATL. With mitogen-activated PBMC from healthy donors, cell cycle analyses revealed that the induction of IRF4 occurred prior to cell cycle progression and the cells that had entered the cell cycle were predominantly IRF4-positive cells. In addition, ectopic expression of IRF4 in Rat-1 fibroblasts increased the S and G2/M phase population significantly. Taken together, our results indicate that IRF4 is involved in the pathogenesis of ATL through its positive effect on the cell cycle, and that IRF4 can be used as a molecular marker of clinical subtype in ATL.

show abstract

Novel approach for simultaneous evaluation of cell phenotype, apoptosis, and cell cycle using multiparameter flow cytometry

et al. 1998

Self Cite

View full text Add to dashboard Cite

Apoptosis is a vital process for organism development and, when disrupted, can lead to abnormalities including cancer and autoimmune diseases. We demonstrate a novel multicolor flow cytometry approach for quantifying apoptosis and cell cycle information of phenotypically distinct populations, using less than 2 × 105 cells per sample. We used incorporation of Cy5‐dUTP into DNA strand breaks by the terminal dUTP nucleotide end labeling (TUNEL) method to determine apoptosis, while cell cycle information was assessed with an ultraviolet DNA binding dye, DAPI. To simultaneously determine surface phenotype, we used paraformaldehyde fixation and a gentle permeabilization protocol combined with FITC‐ and PE‐labeled surface antibodies. Using these fluorochromes, and three‐laser instrumentation, we quantified apoptosis and cell cycle phase in lymphocyte subpopulations from heterogeneous human and murine cell sources, subjected to various culture conditions. Further, we used this method to detect divergent rates of apoptosis in a human, heterogeneous lymphocyte tumor population, demonstrating a potential application for clinical and/or research settings. Thus, we describe a six‐parameter, four‐color flow cytometry approach for evaluating apoptosis and cell cycle with dual surface labels. This method may also be useful as a generalized scheme to assess simultaneously two intracellular targets in a mixed cell population. Cytometry 32:57–65, 1998. © 1998 Wiley‐Liss, Inc.

show abstract

A simplified method for the coordinate examination of apoptosis and surface phenotype of murine lymphocytes

Cited by 38 publications

References 16 publications

A small‐volume technique for simultaneous immunophenotyping and apoptosis detection in rat whole blood by four‐color flow cytometry

A small‐volume technique for simultaneous immunophenotyping and apoptosis detection in rat whole blood by four‐color flow cytometry

Possible Involvement of Interferon Regulatory Factor 4 (IRF4) in a Clinical Subtype of Adult T‐Cell Leukemia

Novel approach for simultaneous evaluation of cell phenotype, apoptosis, and cell cycle using multiparameter flow cytometry

Contact Info

Product

Resources

About