A method to study apoptosis in eosinophils by flow cytometry

The aim of this study was to develop a simple flow cytometric procedure to study eosinophil apoptosis. Eosinophils were isolated from the peripheral b lood of healthy, non-allergic individuals and then cultured in basal cultur e medium. The cells were examined after 24, 48 and 72 h for forward- and si de scatter (FS-SSC) pattern, staining with FDA, PI, and anti-CD95, and ligh t microscopic appearance. After culture for >24 h, two populations with dif ferent FS-SSC-patterns appeared, referred to as A and B. Population A consi sted of living, FDA-positive eosinophils. The eosinophils in population B s howed a lower FS scatter than those in population A and a staining pattern with PI indicating the presence of hypodiploid DNA. Anti-CD95 demonstrated a significant staining of the eosinophils in population B, which increased after 2 days in culture. The cells were sorted using a FAGS-Scan cell sorte r and by Annexin V-coated magnetic beads to permit separate analyses of PI- staining pattern, DNA electrophoresis. and Light microscopic examination of the cells in population B. The present study suggest that it is possible t o discriminate between apoptotic and living eosinophils using the FS-SSC pa ttern and the PI-staining pattern obtained by flow cytometry. (C) 2000 Else vier Science B.V. All rights reserved.