Tests of the ability of mast cells to degranulate. (A) Bcl-2 overexpression but not loss of Apaf-1 or caspase-9 or (B) treatment with the broad-spectrum caspase inhibitor Q-VD allows cytokine-deprived mast cells to retain the ability to degranulate. Mast cell degranulation was determined by staining exocytosing granules with annexin V-FITC. Histograms represent annexin V-FITC fluorescence of PI-negative (viable) cells on the x-axis and relative cell numbers on the y-axis. Mast cells of the indicated genotypes, cultured in the presence or absence (48 hours) of SCF/IL-3, were either left untreated (solid line) or sensitized with IgE anti-TNP (broken line) prior to addition of TNP-BSA to stimulate the Fcϵ receptor and trigger degranulation. FITC-conjugated annexin V was used to stain exocytosing granules of degranulating mast cells and analysis performed on a FACS analyzer, excluding PI-positive (morphologically dead) cells. Note that wild-type mast cells in the absence of SCF/IL-3 are all dead (100% PI positive), and the annexin V-FITC histogram of the dead cell fraction is shown in the inset. The C57 mast cell line served as a positive control for the assay.