Figure 3

(A–D) Dependence on temperature and TRPM2 of forward migration index (FMI) of neutrophil chemotaxis. Neutrophil migration in response to H₂O₂ tracked for 2 h in the ibidi µ-slide chemotaxis system using live-cell time-lapse microscopy. FMI, as a function of temperature, measured with the following genotypes and chemoattractants: (A) WT, to 0.001 µM and 0.01 µM H₂O₂; TRPM2^−/−, to 0.01 µM H₂O₂; (B) WT, to 0.1 nM and 1 nM CXCL2; (C) WT, to 1.0 nM and 10 nM C5a; (D) WT, to 5.0 and 50 ng/ml LPS. Concentrations of conventional chemoattractants giving sub-maximal and near-maximal migration determined in separate experiments (see Supplementary Materials Fig. 3). Each point shows mean ± SEM, n = 3 separate experiments using neutrophils from 3 mice. (E,F) Potentiation of calcium entry through TRPM2 by temperature and H₂O₂. (E) Increase of [Ca]_i in response to a temperature ramp from 37 to 41 °C in neutrophils from WT mice. Threshold for [Ca]_i increase was 40.2 °C in the absence of H₂O₂, 39.1 °C in 1 nM H₂O₂ and 38.8 °C in 10 nM H₂O₂. (F) No increase of [Ca]_i in neutrophils from TRPM2^−/− mice. Traces are from single neutrophils representative of 15–20 cells total in n = 3 separate experiments using neutrophils from 3 mice; all traces had closely similar form. Neutrophil calcium imaging performed in flowing solution in absence of chemotactic gradient (see “Methods” section). Calibration for conversion of F_340/380 ratio to [Ca]_i shown in last panel in G (see “Methods” section). (G,H) Calcium entry elicited by TRPM2-dependent chemoattractants H₂O₂, ADPR and fMLP. (G) Increase of [Ca]_i in response to application of (from left) H₂O₂ (10 µM), ADPR (100 µM) and fMLP (1 µM), in neutrophils from WT mice. In the H₂O₂ experiment (first panel) the [Ca]_i rapidly decreased when external calcium was removed (nominal 0Ca, no EGTA) consistent with Ca entry from the external solution via TRPM2 (Note contracted time scale in this panel). The trace in fMLP shows an example of the method used to convert F_340/380 to [Ca]_i by measuring F_max with exposure to the Ca ionophore ionomycin (10 µM), followed by F_min in 0Ca/2 mM EGTA. (H) No increase of [Ca]_i in neutrophils from TRPM2^−/− mice. Experimental details as in (E,F). All traces from single neutrophils but representative of 15–20 traces from n = 3 separate experiments using 3 mice.