Anti cxcr2 clone 242216

Blood samples of WT and Skap2^−/− mice were taken by heart puncture and depleted of red cells by hypotonic lysis. Cells were incubated with FITC-conjugated anti-Ly6B.2 and APC-conjugated anti-CD45 (clone 30-F11; BD) or PerCP-conjugated anti–Gr-1 and one of the following PE-conjugated antibodies for 30 min on ice in the dark: anti-CD11a (clone M17/4), anti-CD11b (clone M1/80), anti-CD162 (clone 2PH1), anti-CD62L (clone MEL-14), anti-CD44 (clone IM7; all from BD), anti-CXCR2 (clone 242216; R&D Systems), or respective isotype controls (BD). To assess the expression of CD11b and CD18 after stimulation, samples were stimulated for 30 min with 50 ng/ml TNF or left unstimulated in the presence of anti-CD11b (clone M1/80) and anti-CD18 (clone M18/2; BioLegend) antibodies. After fixation, cells were incubated with PE-conjugated secondary antibodies and analyzed by flow cytometry (5,000–10,000 cells per experiment). Neutrophils were gated as CD45^hi Ly6B.2^hi or Gr-1^hi Ly6B.2^hi cells, respectively.

Six hours post-zymosan, CGD peritoneal lavage (PL) cells were plated (1 × 10⁶ cells/well in 24-well plates) and cultured for 24 h to condition media for the transwell assay. Peritoneal exudate was collected 20 h post-zymosan, and MoMacs were enriched via negative selection using a MojoSort Anti-PE Beads kit (BioLegend) and PE-conjugated antibodies to CD19, Ter119, Siglec-F, CD3e, NK1.1, and Ly6G. The MoMacs were plated in 24-well plates above the insert. MoMacs were plated in the presence of the following antagonists or neutralizing antibodies as noted: 10 μg/mL anti-CCL2 (clone 2H5), 10 μg/mL anti-CCL3 (polyclonal, R&D Systems), 10 μg/mL anti-CCL4 (polyclonal, R&D Systems), 5 μg/mL anti-CXCR2 (clone 242216), 10 mg/mL MK-0812 (CCR2 and CCR5), 25 mg/mL BX471 (CCR1), and 100 mM ML339 (CXCR6). Isotype control antibodies were used as controls for each of the neutralizing antibodies at the corresponding concentrations. Vehicle controls (ethanol for MK-0812, DMSO for all others) were used as controls for the antagonist treatments at the corresponding concentrations of vehicle. MoMacs migrating beneath a 5 μm transwell insert (Corning) were counted as noted after plating.