Mab331 100

Anti-CXCR1 (MAB330, R&D Systems), anti-CXCR2 (MAB331-100, R&D Systems), and anti-IL-8 (AB-208-NA, R&D Systems) blocking antibodies were diluted to 50 µg/ml in PBS and added to osteoclast cultures daily.

Arrays of 100 µm diameter clusters of Texas red-labeled zymosan (S. cerevisiae derived bioparticles, ThermoFisher) in microwells were submerged under plasma pretreated with ECU (3 µM) or RA101295 (3 µM) [25]. Human C5-depleted serum (234405, Sigma) was used as the negative control. Isolated neutrophils were suspended in HBSS with 0.5% BSA and pretreated with BLT1/2 antagonists (U-75302 and LY255283, 40 µM, Cayman chemical), and CXCR1/2 (MAB331-100, 2 µg/ml, R&D Systems) and AVA (250 nM) for at least half an hour before being added to each well (500,000 cells/well). Neutrophil swarming around the zymosan clusters was monitored for 8 hours by time-lapse imaging (one image every 2 minutes).