Flow cytometric procedure to detect apoptosis of bovine polymorphonuclear leukocytes in blood.
A flow cytometric technique was used to detect apoptosis and necrosis of bovine polymorphonuclear neutrophil leukocytes (PMN) using fluorescein isothiocyanate labeled annexin-V and propidium iodide (PI). Isolation of PMN from the blood following lysis by water or NH4Cl resulted in false positive results for apoptosis. Therefore, a method was developed to identify living, apoptotic and necrotic PMN simultaneously in a single 100 microl blood sample. To establish a positive control for PMN apoptosis, the effect of cycloheximide, actinomycin D, diamide, buthionine sulfoximine and sodium arsenite, that have been described to induce apoptosis by various mechanisms was tested. Only actinomycin D induced a significant increase in the percentage of apoptotic PMN after 2 h. Incubation of blood for 6 h with cycloheximide, actinomycin D and buthionine sulfoximine resulted in a significant increase of apoptotic PMN compared to control values. Sodium arsenite, mainly caused necrosis after 6 h of incubation.
Van Oostveldt
K
K
University of Ghent, Faculty of Veterinary Medicine, Department of Physiology, Biochemistry and Biometrics, Merelbeke, Belgium.
Dosogne
H
H
Burvenich
C
C
Paape
M J
MJ
Brochez
V
V
Van den Eeckhout
E
E
D000818:Animals; D017209:Apoptosis; D002417:Cattle / Q000097:blood*; D002469:Cell Separation / Q000662:veterinary*; D005260:Female; D005434:Flow Cytometry / Q000662:veterinary*; D016650:Fluorescein-5-isothiocyanate; D009504:Neutrophils / Q000166:cytology*
D016650:Fluorescein-5-isothiocyanate