ASM activation and ceramide generation in neutrophil apoptosis. (A) Freshly isolated human neutrophils and neutrophils cultured for 4 and 6 hours were lysed and assayed for ceramide content (picomoles per million cells) using a DAG kinase assay. (B) Neutrophils were isolated from C57BL6 wild-type (+/+) and ASM knockout (–/–) mice, and ceramide content was determined after 0 hours (□) and 6 hours (▪) of culture. (C) Human neutrophils were incubated for 0 hours or 6 hours, lysed, and the heavy membrane (HM), cytosol (Cyt), and plasma membrane (PM) fractions isolated by differential centrifugation and probed for ASM by Western blotting. A representative of 3 experiments is shown. Neutrophils were isolated from C57BL6 wild-type (+/+) and ASM knockout (–/–) mice and cultured for 20 hours. Apoptosis was measured by loss of DiOC₆ staining assessed by FACS; open area indicates DiOC₆ low cells and shaded area indicates DiOC₆ high cells (D) and expressed as the percentage of apoptotic cells (E). For panels B and E data are mean ± SD of 4 mice.