Fig. 1.

Calcium ionophores induce rapid NETosis. (A) NET release in response to A23187 or PMA was measured using a plate reader assay (n = 5). A23187- and PMA-mediated NETosis in human neutrophils follow different kinetics of NET release. Ionophore-induced NETosis is faster than that of PMA-induced NETosis. (B and C) Neutrophils were activated with A23187 (B) or ionomycin (C) in the presence or absence of calcium. NETosis was measured using a plate reader assay. NET release is expressed as percentage of total DNA (n = 3). Calcium ionophore-induced NETosis requires extracellular calcium. (D) Neutrophils were incubated in the presence or absence of A23187 (4 µM) or ionomycin (5 µM) for 300 min. Cells were stained for DNA (green) and MPO (red). Immunofluorescence imaging shows that the calcium ionophore A23187 and ionomycin induce NETosis. (Scale bar, 10 µm.) Images are representative of three independent experiments. A23, A23187; Ca²⁺, calcium chloride; Io, ionomycin. *P < 0.05; **P < 0.01; ***P < 0.001 (A–C, two-way ANOVA).