(A–D) Histone H3 was citrullinated during NETosis in response to all stimuli bar PMA. (A) Primary neutrophils were stimulated for 90 min with PMA, A23187, nigericin, C. albicans or GBS, lysed and assayed for the presence of citrullinated histone H3 and GAPDH by SDS-PAGE electrophoresis and Western immunoblotting. (B–D) NETosis rate and percentage of citrullinated cells in response to (B) PMA, (C) A23187 or nigericin and (D) C. albicans or GBS. Graphs show mean ± SD from a representative of two independent experiments. (E–G) NETosis rate in neutrophils pre-treated with the PAD inhibitor Cl-amidine at 200 µM, BB-Cl-amidine at 10 µM, TDFA at 200 µM, or DMSO as control and stimulated with (E) PMA, (F) A23187 or nigericin and (G) C. albicans or GBS and analysed for NET production by immunofluorescence. Graphs show mean ± SEM from 10 independent experiments. *p<0.05, NS = not significant.