A new four-color flow cytometric assay to detect apoptosis in lymphocyte subsets of cultured peripheral blood cells

Abstract

Background Human peripheral blood lymphocytes kept in culture after isolation die by an apoptotic process. Detection of apoptosis with labeled Annexin V to demonstrate loss of plasma membrane asymmetry is sensitive, specific, and easy using flow cytometry. This is true in lymphoblastic cell lines when combining Annexin V‐fluorescein isothiocyanate (FITC) and propidium iodide (PI). However, measurement of apoptosis by flow cytometry in isolated human lymphocytes using Annexin V‐FITC/PI is disturbed by the presence of a variable percentage of erythrocytes in the isolated lymphocyte population. To overcome this problem, we have developed and tested a new four‐color flow cytometric assay to detect apoptosis in lymphocyte subsets of cultured peripheral blood cells. Methods Peripheral blood lymphocytes are isolated by density gradient centrifugation. Nucleus‐containing cells are selected using CD45‐phycoerythrin (PE). The lymphocyte subset of interest is selected using CD4, CD8, or CD19 energy‐coupled dye (ECD) labeling. Apoptosis is detected using Annexin V‐FITC with 7‐amino‐Actinomycin‐D (7‐AAD) to distinguish early apoptotic from late apoptotic lymphocytes. Results We have developed a new technique to detect apoptosis in isolated human peripheral blood lymphocyte subsets with good reproducibility, coefficient of variation < 17%. Conclusions We now have a validated tool to study apoptosis in subsets of isolated human lymphocytes to increase our knowledge of pathogenesis and therapies in lymphoreticular malignancies. Cytometry 40:167–171, 2000 © 2000 Wiley‐Liss, Inc.

Regulation of Cu/Zn-Superoxide Dismutase Expression via the Phosphatidylinositol 3 Kinase/Akt Pathway and Nuclear Factor-κB

“…This alteration was analyzed with Annexin-V-PE. To discriminate apoptosis from necrosis, we stained the cells simultaneously with the red fluorescent vital dye 7-AAD, which enters cells lacking membrane integrity and, therefore, allows for detection of necrotic cells (Hasper et al, 2000). EGFP and myr-EGFP-Akt1 cells were maintained under low-serum conditions for 16 hr and then were submitted to 0.5 or 1.5 mM H 2 O 2 for 6 hr and finally were stained for 15 min with Annexin-V-PE and 7-AAD.…”

Hematopoietic Stem Cell Injury Induced by Ionizing Radiation

“…Third, HSCs from p53-deficient mice are less sensitive to IR than are those from wild-type mice (29, 63,86) and treatment with a p53 inhibitor protected mice from IRinduced lethal damage by suppression of p53-dependent apoptosis (81). In addition, using flow cytometric analysis of Annexin V and/or 7-AAD staining in LSK cell population enriched with HSCs (56,122), we demonstrated that IRinduced cell death in LSK cells mainly by apoptosis but not by necrosis (108,109).…”

The phytoestrogen genistein induces thymic and immune changes: A human health concern?

“…iodide (PI) by using the annexin-V-FLUOS staining kit (Roche Diagnostics), then apoptosis was quantitated by flow cytometry (11). Annexin staining measures cell membrane phophatidylserine externalization, which is a marker of the earlier stages of apoptosis.…”

“…We therefore treated mice with DMSO, E 2 , or 80 mg͞kg per day genistein for 7 days, and then quantitated apoptosis (11). The 7-day genistein treatment decreased thymic weight 62%, only slightly less than the 73% decrease after 21 days of treatment with this genistein dose, indicating genistein effects occur rapidly.…”