Anti ccl3

Mice were euthanized by CO₂ asphyxiation. Peritoneal lavage was performed by using 8 mL lavage buffer (Hanks balanced salt solution, 10 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, 1 mM ethylenediaminetetraacetic acid). Cells were passed through a 70 μm strainer to ensure a single cell suspension. Cells were counted, resuspended in culture medium (Roswell Park Memorial Institute 1640 Medium supplemented with 10% fetal bovine serum, 100 U/mL of penicillin, 100 U/mL of streptomycin, and 29.2 mg/mL of L-glutamine), and plated at a density of 1 × 10⁶ cells/well in 24-well plates. Where indicated, cells were plated in the presence of the following antagonists or antibodies at the time of plating: 8 μg/mL U-75302, 83.3 μM LY255283, 5 μM reparixin L-lysine, 1 μM Boc-MLF, 1 μM cyclosporin H, 1 μM WRW4, 10 μg/mL anti-CD11b (clone 5C6), 10 μg/mL anti-CCL2 (clone 2H5), 10 μg/mL anti CCL3 (polyclonal, R&D Systems), and 10 μg/mL anti-CCL4 (polyclonal, R&D Systems). Isotype control antibodies were used as controls for each of the neutralizing antibodies at the corresponding concentrations. Vehicle controls (ethanol for U-75302, DMSO for LY255283, reparixin L-lysine, Boc-MLF, and cyclosporin H, and water for WRW4) were used as controls for the antagonist treatments at the corresponding concentrations of vehicle. Cells were cultured at 37°C with 5% CO₂ for up to 24 h.

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.