Stim1^−/−neutrophils are defective in superoxide production. Superoxide production by WT and stim1^−/− neutrophils was measured by luminol-based chemiluminescence in the presence (left panels) or absence (right panels) of extracellular calcium. (A) WT and stim1^−/− neutrophils (2 × 10⁵ cells per well) were plated in 96-well plates precoated with 0.5% milk (to block adhesion) in the presence of fMLF (10 μM), and luminescence was monitored in a Spectramax plate reader at 37°C over the indicated time period. Integrin-dependent ROS production was measured in plates coated with 15 μg/mL pRGD (B) or 150 μg/mL fibrinogen in the presence of 10 ng/mL TNF-α (C). The intracellular calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid AM (10 μM) was added as indicated. FcγR-generated superoxide was measured by incubating WT and stim1^−/− neutrophils with IgG-opsonized zymosan (D) or SRBCs with TNF-α priming (10 ng/mL) (E). Luminescence was monitored over the indicated time period. Data are mean (± standard deviation; n = 3 wells each) and are representative of at least 3 independent experiments. Statistical analysis at peak response was P < .001 between WT and stim1^−/− cells in the presence of calcium.