zVAD sensitizes neutrophils to TNF-α–induced cell death in a dose-dependent manner.

(A-B) Neutrophils were preincubated with or without zVAD at the indicated concentrations for 1 hour. At time 0, TNF-α was supplemented to the final concentrations shown. Cell death was analyzed at 6 hours by dual-parameter flow cytometry utilizing FITC-conjugated annexin V (annexin V–FITC), which binds specifically to phosphatidylserine on the cell membrane and marks early apoptotic, primary necrotic, and late apoptotic/secondary necrotic cells, and propidium iodide (PI), which enters dead cells with breached membrane integrity and marks primary and late apoptotic/secondary necrotic cells. Each panel represents data collected from 10 000 total events. In each panel, the left lower quadrant represents remaining live cells that do not bind annexin V–FITC and exclude PI. The right lower quadrant represents the accumulation of early apoptotic cells that have externalized membrane phosphatidylserine but still retain membrane integrity. The right upper quadrant represents the accumulation of both late apoptotic cells that have lost membrane integrity (secondary necrosis) and primary necrotic cells. The percentages of cells in each of these quadrants are indicated. (C) Neutrophils preincubated with or without zVAD (100 μM) for 1 hour were incubated in the absence or presence of TNF-α (10 ng/mL) for 3 additional hours and subsequently lysed and subcellularly fractionated (see “Materials and methods”). Accumulation of cytosolic cytochrome c (15 kDa) in the mitochondria-poor fraction was assessed by immunoblotting. Results shown above are representative of 3 independent experiments.