Validation of Salsa6f and of myeloid Stim1/2 ablation. (A) Flow cytometry gating strategy to isolate tdT⁺Ly6G⁺CD115⁻F4/80⁻ neutrophils from flushed mouse BM. The two FACS profiles at bottom left are replicated in Fig. 1 A. Cartoon shows the LSL-Salsa6f cassette with its Ca²⁺-insensitive tdTomato (tdT⁺) and Ca²⁺-sensitive GCaMP6f inserted at the Rosa26 locus. (B) Percentage of BM neutrophils isolated by negative selection from mice expressing or not Salsa6f. n = 6 pairs of mice, two-tailed Welch’s t test. (C) Simultaneous Fura-2 and Salsa6f recordings in cells exposed to increasing extracellular [Ca²⁺] in the presence of ionomycin (left) and steady-state Fura-2 and Salsa6f ratios as a function of intracellular [Ca²⁺], calculated from [Ca²⁺]_ext using the Kd of Fura2 (right). (D) TIRF kymograph of Ca²⁺ waves propagating in an adherent neutrophil (top) and changes in GCaMP6f intensity in three aligned regions (bottom, regions are indicated by dotted circles on the TIRF image) separated by 3.6 µm along the kymograph axis (dotted line). See Video 2. Representative of 10 recordings. (E) Flow cytometry Fluo-8 recordings and quantification of Ca²⁺ responses evoked by Ca²⁺ readmission to WT and Stim1/2^−/− neutrophils exposed to Tg (1 µM) in Ca²⁺-free medium. n = 3 recordings from one pair of mice. (F) Salsa6f recordings and quantification of the Tg-Ca²⁺ evoked responses in WT and Stim1/2^−/− neutrophils. n = 2 recordings from two pairs of mice. AUC, area under the curve. (G) Luminometric LO-12 recordings of the ROS production evoked by PMA in WT and Stim1/2^−/− neutrophils. n = 5/6 mice, in triplicate recordings.