Effect of Stim1/2 ablation on Ca ²⁺ handling proteins and actin dynamics. (A) Representative micrographs of PLAs between the indicated molecular targets in WT and Stim1/2-deficient neutrophils and in the absence of primary antibodies (left, scale bars: 10 µm) and quantification of the interactions (right, n = 11/10, 10/10 and 3/3 micrographs with >10 cells for each condition from 2/2/1 mice pairs, Mann–Whitney U test). (B) Confocal micrographs of WT and Stim1/2^−/− neutrophils plated on PLL for 30 min, fixed and stained with an anti-PMCA antibody (left, nuclei stained blue with DAPI) and averaged cell-associated fluorescence area (right, n = 32/27 cells from one mice pair, Student’s unpaired t test). (C) Basal Salsa6f ratio of WT and Stim1/2-deficient neutrophils recorded with two different imaging cameras. n = 108/236 and 37/87 cells from three mice pairs. Lines show median values, larger symbols mouse average. Data from camera 1 were converted to [Ca²⁺]_cyt in Fig. 6 B. (D) Proportion of WT neutrophils exhibiting Ca²⁺ transients before and after switching from 2 to 10 mM [Ca²⁺]_ext (left) and quantification of the spreading area of WT neutrophils adhered in 2 and 10 mM [Ca²⁺]_ext (right, n = 22/33 cells in 4/5 recordings from two WT/Stim1/2^−/− mice pairs, exact two-tailed Mann–Whitney U test on cell values).