Flow cytometric procedure to detect apoptosis of bovine polymorphonuclear leukocytes in blood

A flow cytometric technique was used to detect apoptosis and necrosis of bo vine polymorphonuclear neutrophil leukocytes (PMN) using fluorescein isothi ocyanate labeled annexin-V and propidium iodide (PI). Isolation of PMN from the blood following lysis by water or NH4Cl resulted in false positive res ults for apoptosis. Therefore, a method was developed to identify living, a poptotic and necrotic PMN simultaneously in a single 100 mu l blood sample. To establish a positive control for PMN apoptosis, the effect of cyclohexi mide, actinomycin D, diamide, buthionine sulfoximine and sodium arsenite, t hat have been described to induce apoptosis by various mechanisms was teste d. Only actinomycin D induced a significant increase in the percentage of a poptotic PMN after 2 h. Incubation of blood for 6 h with cycloheximide, act inomycin D and buthionine sulfoximine resulted in a significant increase of apoptotic PMN compared to control values. Sodium arsenite, mainly caused n ecrosis after 6 h of incubation. (C) 1999 Elsevier Science B.V. All rights reserved.