Figure 4.

Neutrophil cytokine synthesis requires STIM2. (A-C) Analysis of cytokine production in whole bone marrow cells (1 × 10⁶) from WT, Stim1^VavCre, Stim2^VavCre, and Stim1/2^VavCre mice incubated with (A) zymosan (100 μg/ml), (B) PAM3CSK (1 μg/mL), or (C) insoluble immune complexes (10 μg/mL) in RPMI ([Ca²⁺] 0.7 mM) for 5 hours. Neutrophils were identified by double staining for Ly-6G and CD11b, and cytokine production was measured by intracellular staining for TNFα, IL-10, and IFN-γ. (A) FACS plots (left) are representative of 3 independent experiments, and bar graphs (right) represent mean ± SEM compiled from multiple experiments. (D) Analysis of cytokine transcripts by RT-qPCR. Neutrophils were purified from WT, Stim1^VavCre, Stim2^VavCre, and Stim1/2^VavCre mice by negative selection yielding more than 95% pure neutrophils. Cells were stimulated with zymosan (100 μg/mL) for 4 hours, and RNA transcript levels of the indicated cytokines measured by RT-qPCR. Mean ± SEM compiled from 3 independent experiments. #P < .05.