G-CSF inhibits calpain activation, upstream of caspase-3. To determine calpain and caspase-3 activation during neutrophil apoptosis, cytosolic extracts of neutrophils, incubated in the presence of 10 ng/mL G-CSF and various inhibitors as indicated, were made at the indicated times (A,B) or after 8 hours (C). Protease activity was determined by incubating the samples in the presence of a fluorescent peptide, specific for either caspase-3 or calpains. After incubation, the increase in fluorescence was used as an indicator of the relative protease activity in the samples. To control for inhibitor/substrate specificity, the sample with the highest protease activity (the 8-hour control) was treated with 20 μM of the pan-caspase inhibitor z-VAD or 20 μM of the calpain inhibitor 3 (CI3). Activity is expressed as a relative to the control (8 hours, no inhibitors) as corrected for background activity (0 hours, no inhibitors). All data represent the mean (± SEM) of 3 independent experiments performed in duplicate. Significance was determined relative to the control (8 hours, no inhibitors) by paired, 1-tailed Student t test. *P < .05, **P < .001, and n.s. (not significant), P > .05.