Figure 5

Effect of pharmacological blockers on neutrophil migration towards H₂O₂ and chemokine CXCL2. (A) Gradient of H₂O₂ (10 μM over 1 mm). From top: H₂O₂ alone; TRPM2 blocker ACA (N-(p-amylcinnamoyl)anthranilic acid, 10 μM) completely inhibits both migration and Ca pulses (reproduced from Fig. 4); migration is slowed and Ca²⁺ pulses partially inhibited by the Src-family kinase inhibitor PP2 (10 µM), PI3kinase inhibitor wortmannin (100 nM) and Rac inhibitor EHT 1864 (50 µM). Images shown at 10 s intervals in each panel. Long blue arrows—movement of neutrophil a distance equal to or greater than the cell diameter; arrowheads—movement less than cell diameter; no arrow—no significant movement. Other details as in Fig. 4. Images representative of n = 3 individual experiments. (B) Similar experiment with gradient of CXCL2 (10 nM). Migration and Ca²⁺ pulses are unaffected by ACA (10 µM) and PP2(10 µM), while migration is slowed and Ca²⁺ pulse amplitude reduced by wortmannin (100 nM) and ETH 1864 (50 µM). Images representative of n = 3 individual experiments. (C) Peak calcium and cell velocity for the conditions shown in (A). Bars show average of n = 3 individual cells. Two-way ANOVA and Tukey–Kramer post-hoc test compared to first bar. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001, ns = not significant. Other details as in Fig. 4. (D) Similar plots for conditions shown in (B). Similar experiments for conventional chemoattractants C5a and LPS shown in Supplementary Materials Fig. 5.