Effect of Stim1/2 ablation on IP ₃ R expression and basal [Ca ²⁺ ] _cyt levels and effect of Ca ²⁺ supplementation on Ca ²⁺ activity and spreading of Stim1/2-deficient neutrophils. (A) Representative z-stack maximal intensity projection micrographs of PLAs between IP₃R3 and the PM Ca²⁺ pump PMCA in WT and Stim1/2-deficient purified neutrophils (left, red dots on the confocal micrographs, nuclei stained blue with DAPI, scale bars: 10 µm) and quantification of the interactions (right, n = 10/10 micrographs with >10 cells for each condition from two mice pairs, Mann–Whitney U test). (B) Western blot showing IP₃R1 protein levels in WT and Stim1/2-deficient purified neutrophils. (C) Representative immuno-electron micrographs (left) of WT and Stim1/2-deficient purified neutrophils exposed to an anti-IP₃R1 antibody coupled to 10-nm-diameter gold particles (arrows) and quantification of the number of gold particles in the cytosol and in regions <50 nm from the PM in WT and Stim1/2^KO neutrophils (right, n = 20/20 cells from one pair of mice, exact two-tailed Mann–Whitney U test. Scale bar: 500 nm). (D) Resting cytosolic Ca²⁺ concentration of WT and Stim1/2-deficient flushed neutrophils, measured with Salsa6f in non-spiking cells. Salsa6f ratios were converted to [Ca²⁺]_cyt using the calibration curve of Fig. 1 G. n = 108/230 cells from two pairs of mice, unpaired two-tailed t test on cell values, lines are median values, larger symbols mouse average. (E) Salsa6f recordings (left), basal Ca²⁺ levels (middle), and proportion of flushed Stim1/2^KO neutrophils exhibiting Ca²⁺ transients before and after switching from 2 to 10 mM [Ca²⁺]_ext (right, n = 151 cells in eight recordings from two pairs of mice, Student’s paired two-tailed t test). (F) Change in the size of CellMask TIRF footprints of flushed Stim1/2^KO neutrophils adhering to PLL-coated glass in 2 and 10 mM [Ca²⁺]_ext (left) and quantification of the spreading area (right, n = 22/33 cells in 4/5 recordings from 2 WT/Stim1/2^KO mice pairs, exact two-tailed Mann–Whitney U test on cell values). Source data are available for this figure: SourceData F6.