The neutrophil chemotaxis imaging assays were performed as previously described (Zengel et al., 2011 (link); Bzymek et al., 2016 (link)). Briefly, 6 µl of 3 × 106 cells/ml neutrophils were allowed to adhere for 20 min to cell channels connecting two 60-µl reservoirs on µ-Slide Chemotaxis chambers (80326; Ibidi). Two reservoirs were gently filled with 60 µl media, and reservoirs were loaded with 30 µl of 1 µM LTB4 or PBS. Cells were imaged by phase-contrast microscopy (2.4 Nikon Live) via a 10× objective lens. Images were captured every 1 min for 1 h, and cell migration tracks were analyzed with ImageJ (NIH) using a manual track plugin and chemotaxis and migration tools from Ibidi. 40 randomly selected neutrophils were manually tracked in each chemotaxis exp. FMI, directness, distance, and velocity were used as the metrics for chemotactic efficiency.