(a)
(b)
Figure 3: Neutrophil hypercitrullination requires extracellular calcium. (a) Freshly isolated neutrophils were cultured in medium with 2 mM CaCl2 alone or in medium with the indicated concentrations of calcium and 1 μM ionomycin for 30 min. Citrullination was detected with the clone F95 antibody (upper panel) and equal loading verified with anti-β-actin (lower panel). (b) Neutrophils were incubated in medium with 2 mM CaCl2 (left panel) or with 1 mM EDTA (right panel) alone for 0 or 4 hours or with 1 μM ionomycin for 4 h. Citrullination was detected with the F95 antibody (upper panel) and equal loading verified with anti-β-actin (lower panel). Results are shown from one donor representative of three different donors.