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 blood of healthy, non-allergic individuals and then cultured in basal culture medium. The cells were examined after 24, 48 and 72 h for forward- and side scatter (FS-SSC) pattern, staining with FDA, PI, and anti-CD95, and light microscopic appearance. After culture for >24 h, two populations with different FS-SSC-patterns appeared, referred to as A and B. Population A consisted of living, FDA-positive eosinophils. The eosinophils in population B showed 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 FACS-Scan cell sorter 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 to discriminate between apoptotic and living eosinophils using the FS-SSC pattern and the PI-staining pattern obtained by flow cytometry.
Sandström
K
K
Department of Medical Sciences, Clinical Chemistry, Uppsala University, Sweden. kristina.sandstrom@medsci.uu.se
Håkansson
L
L
Lukinius
A
A
Venge
P
P
D017304:Annexin A5 / Q000302:isolation & purification; D000943:Antigens, Differentiation; D017209:Apoptosis; D002478:Cells, Cultured; D053938:DNA Fragmentation; D004804:Eosinophils / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008027:Light; D009336:Necrosis; D010718:Phosphatidylserines / Q000302:isolation & purification; D011419:Propidium; D012542:Scattering, Radiation; D019810:Sodium Azide / Q000494:pharmacology; D013194:Staining and Labeling; D019014:fas Receptor / Q000302:isolation & purification
D017304:Annexin A5; D000943:Antigens, Differentiation; D010718:Phosphatidylserines; D019014:fas Receptor; D011419:Propidium; D019810:Sodium Azide