Neutrophil Chemotaxis Assay

Human neutrophils were isolated from peripheral whole blood collected from healthy volunteers using a Ficoll-Histopaque density gradient. Red blood cells were lysed with hypotonic buffer and isolated neutrophils were resuspended in Hank’s Balanced Salt Solution (HBSS, Gibco life technologies™). Neutrophils (3 × 10⁶ cells/mL) were placed on Ibidi chemotaxis chambers following manufacturer’s protocol and as described earlier [27 (link)] (μ-Slide Chemotaxis 80326, Ibidi cells in focus, Germany). Chemotaxis toward gradient of LTB₄, RvD1, RvD2, RvD3, RvD4, RvD5, 17-oxo-RvD4, 10-trans-RvD4, or 10,13-trans-RvD4 (10 nM each) or vehicle (HBSS containing 0.1% ethanol) was then recorded for a 30-minute period with a digital monochrome/color switching camera using BZ-II Viewer software (Keyence). Cell migration was analyzed using the cell-tracking function in ImageJ software. At least 60 cells per condition were analyzed from each donor for four separate donors.