Anti ly 6g clone 1a8

Aliquots of 50 µl of single cell suspensions from each sample were dispensed into 96-well round bottom microtiter plates and stained with 1∶200 dilutions of antibodies (BD Pharmingen or eBioscience): anti-Ly-6G (clone 1A8, FITC labeled), anti-CD11b (clone M1/70, labeled with phycoerythrin-Cy7), and anti-F4/80 (clone BM8, allophycocyanin labeled). Rat IgG2a and IgG2b were used as isotype controls. Cells were stained for 30 min at 4°C, spun at 650× g for 1 min and fixed with IC Fixation Buffer (eBioscience, San Diego, CA) for 1 h at 4°C. Cells were spun at 650× g for 1 min and resuspended in PBS with 1% fetal bovine serum. Cell phenotype data were acquired on a Partec CyFlow ML flow cytometer and analyzed with FloMax (Partec) and FloJo (Tree Star) software. Gating strategies were as previously described [31] (link). Neutrophils were defined as Ly-6G⁺F4/80⁻. Neutrophils that expressed high levels of CD11b [29] (link) (CD11b^high) were defined as activated neutrophils [31] (link)–[35] (link). Macrophages were defined as F4/80⁺Ly6G⁻ cells.

After the removal of the left lobe of mice lung, the tissue was chopped and digested with 15 mg/mL collagenase (Type D; Roche Diagnostics) and 25 μg/mL DNase (Type 1; Roche Diagnostics) in 4 mL RPMI + 10% CFS for 1 h at 37 °C with agitation. Lung was then passed through a 70 μm sieve (BD Bioscience). The resulting cell suspension was washed, red blood cells were lysed, and the remaining cells were resuspended in 1 mL RPMI. To analyze cell populations, the cell suspension was labeled using monoclonal antibodies anti-CD45 (clone 30-F11) (1:100) anti-Ly6G (clone 1A8) (1:400), anti-F4/80 (clone BM8) (1:100), anti-CCR5 (HM-CCR5 (7A4)) (1:200) from eBioscience for 30 min. All data was acquired by flow cytometry (FACSCalibur; BD Biosciences PharMingen) and analyzed using FlowJo software (Tree Star, Inc., Ashland, OR, USA). Gating strategies for the identification of neutrophils and macrophages in these experiments are shown in Figure S2.