Neutrophil Depletion in Pneumonia Mouse Model

We used c57BL/6 mice (Taconic) aged 7 to 9 weeks for all mouse experiments, and pulmonary infection was performed as previously (14 (link), 67 (link)). At 48 and 4 h prior to the procedure, mice were injected intraperitoneally with 225 μg of rat anti-mouse Ly6G (1A8) or a control rat anti-mouse IgG_2a (2A3) (BioXcell). Neutrophil depletion was confirmed previously using flow cytometry of lung homogenates (Ly6G⁺, Ly6C⁻, CD11b⁻). Inocula were prepared by resuspending MMC39 in a Tris-glycine buffer containing 5 mg/ml of an ovalbumin control mIgG₁ (Crown Biosciences), 17H12 mIgG₁, or 17H12 mIgG₃ to a final concentration of 6 × 10⁶ or 3 × 10⁷ CFU/ml. After 1 h of opsonization, 50 μl of the inoculum was instilled into the surgically exposed trachea of a mouse under ketamine/xylazine using a bent 27-gauge needle. After 20 h, mice were euthanized, and lungs, liver, and spleen were collected and processed in NP-40 or PBS and diluted to enumerate CFU. Supernatants of lung homogenates used for cytokine analysis were stored at −80°C with 1× Pierce proteinase inhibitor until testing using Bio-Plex Pro mouse cytokine Th17 panel A with additional GM-CSF and IL-12p70 singleplex sets on a Bio-Plex 200 Platform (Bio-Rad). Cytokine levels were normalized against total protein measured by Bradford assay.