Analysis of cytochrome c release in cytokine-deprived mast cells. Cytokine deprivation causes release of cytochrome c from mitochondria in mast cells, and this can be blocked by (A) Bcl-2 overexpression but not by loss of Apaf-1 or caspase-9 or (B) by addition of the broad-spectrum caspase inhibitor Q-VD. Mast cells (wild-type, vav-Bcl2 transgenic, Apaf-1^-/-, or caspase-9^-/-) were either cultured in IL-3 plus SCF or deprived of cytokines for 48 hours. In panel B, wild-type cells were cultured in the presence of the broad-spectrum caspase inhibitor Q-VD. Mitochondrial cytochrome c release was measured by permeabilizing cells, followed by fixation and immunofluorescent staining with antibodies to cytochrome c. In cells with damaged mitochondria, cytochrome c will be washed out, resulting in low-intensity anti-cytochrome c staining, whereas cells with intact outer mitochondrial membranes will show high-intensity staining in this assay. (C) Mast cells (wt) were cultured for 48 hours in SCF plus IL-3 or deprived of both cytokines, either in the presence or absence of the caspase inhibitor Q-VD (50 μM). Cells were then stained with PI plus FITC-conjugated annexin V and analyzed by flow cytometry.