Neutrophils of P-selectin^ΔCT/ΔCT mice are primed to release NETs in vitro. Neutrophils isolated from peripheral blood of WT and P-selectin^ΔCT/ΔCT (ΔCT/ΔCT) mice were kept unstimulated (Unst) or stimulated with PAF (A), ionomycin (iono) (B), or PMA (C). The percentage of H3Cit⁺ neutrophils and NET release were quantified (n = 4; *P < .05, **P < .01, ***P < .001). (D) Representative fluorescence microscopy images of unstimulated or ionomycin-stimulated cells showing H3Cit-negative neutrophils (yellow arrow), H3Cit⁺ neutrophils without NET release (red arrow), and H3Cit⁺ neutrophils releasing NETs (white arrowhead). Scale bar represents 20 μm. (E) Left: Neutrophils isolated from peripheral blood of P-selectin^−/−, WT, or P-selectin^ΔCT/ΔCT mice were double stained with APC-conjugated anti-Ly6G and FITC-conjugated anti-P-selectin, and the mean fluorescence intensity (MFI) of P-selectin-positive cells was analyzed in the neutrophil gate (Ly6G-positive cells) by flow cytometry (n = 4-5; *P < .05). Right: Representative fluorescence-activated cell sorter plots for each genotype.