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

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