Caspase-dependent apoptosis of Kostmann bone marrow progenitor cells. (A) CD34⁺ cells from patient 2 were incubated overnight in the presence or absence of the caspase inhibitor zVAD-fmk (10 μM) and subsequently labeled with annexin V to allow for the detection of apoptosis. Cells incubated in the presence of anti-Fas antibodies (250 ng/mL) were also tested. For comparison, data obtained in a healthy control are also shown. (B) Caspase-3-like enzyme activity in bone marrow progenitor cells from Kostmann patients. CD34⁺ cells isolated from patients 1 and 5 were incubated overnight in the presence or absence of zVAD-fmk (10 μM), and lysates were then assessed for DEVD-AMC cleavage in a continuous fluorometric assay. The maximum linear rate of AMC release (picomoles per minute) was estimated by linear regression (r² > 0.99). Cells from patient 1 were also evaluated after treatment with anti-Fas antibodies (250 ng/mL). Data obtained in cells from healthy controls are shown for comparison (mean ± SD; n = 3). All patient samples were obtained 12 hours after the last dose of G-CSF.