Ca ²⁺ elevations reported by Salsa6f in adherent murine neutrophils. (A) Representative micrograph and flow cytometry profiles of BM cells from CEBPa-Salsa6f mice. Myeloid Salsa6f⁺ cells have a high tdT fluorescence (red) and most express the granulocyte marker Ly6G (cyan). (B) Proportion of Ly6G⁺ cells in sorted and adhered TdT⁺ BM cells (left, n = 7/54, Student’s unpaired two-tailed t test) and proportion of confirmed neutrophils (CD115⁻F4/80⁻) in TdT⁺Ly6G⁺ cells purified with a neutrophil kit or flushed from BM (right, n = 5/7, Student’s unpaired two-tailed t test, cells termed “purified” and “flushed” thereafter). (C) Representative micrographs of the Ca²⁺ fluctuations reported by Salsa6f in purified neutrophils plated on PLL. Ca²⁺ elevations increase the green fluorescence of GCamP6f. See Video 1. (D) Representative Ca²⁺ recordings of flushed murine neutrophils crawling on PLL (left) and incidence and frequency of Ca²⁺ spikes. (right, n = 25 recordings from three mice). (E) Ca²⁺ fluctuations recorded by Fura-2 and Salsa6f in purified neutrophils loaded or not with Fura-2 (left) and kinetic properties of the Ca²⁺ transients recorded in each condition (right, n = 49/90/122 cells from three mice). (F) Representative micrographs of Fura-2–loaded Salsa6f⁺-purified neutrophils, showing the cellular distribution of the TdT and Fura-2 fluorescence. (G) In situ calibration of Salsa6f and Fura-2 in cells equilibrated at increasing extracellular [Ca²⁺] with ionomycin. n = 3 mice.