A two-stage process in gradient sensing is observed in endogenously motile neutrophils. In support of Figs. 2 and 3. (A and B) Example of time evolution of Lifeact polarity (red color) with neutrophil speed (blue color; A) and cosine δ (green color; B) from two individual neutrophils. LW: time of laser wounding. (C) Examples of neutrophil Lifeact distribution with an indication of cell speed (u) and Lifeact polarity (l.p.) at the same timepoint, for mechanical wounding. Arrows indicate the direction of motion. Dashed lines indicate the automated separation of the front (F) and rear (R) part of cell. Scale bar = 5 μm. (D) Temporal cross-correlation between Lifeact polarity and speed post–mechanical wounding. Average from n = 14 migrating cells, from 2 larvae. Mean and SEM are shown. (E) Scheme of zebrafish larva with indication of laser wounding area (red dot) and imaging area (blue dashed square). Time-lapse sequence of two-photon confocal image projections showing a neutrophil (white) in a Tg(mpx:Lifeact-Ruby) zebrafish larva, at 0.5 min pre–laser wounding and at 1 and 5 min post–laser wounding. White arrows indicate the speed vector. Red arrows indicate the side of cell with higher Lifeact abundance. Scale bar = 10 μm. (F and G) Neutrophil Lifeact polarity, speed, cosine of θ, and cosine of δ, in relation to time, respectively. Time sequence was synchronized based on time of the LW (F) or the time that each individual neutrophil beginning of movement post–laser wounding (G). (D)n = 21 cells, from 9 larvae. (E)n = 18 cells, from 9 larvae.