Fitc anti mouse ly 6g ly 6c gr 1

Single cells were obtained from lungs digested by collagenase. The cells were stained for 1 h with abs PE Rat Anti-Mouse F4/80 (BD Pharmingen Cat# 565410), PE/Cy7 Anti-Mouse/Human CD11b (BioLegend Cat# 101215), PerCP/Cyanine5.5 Anti-Mouse CD45 (BioLegend Cat# 103132), and FITC Anti-Mouse Ly-6G/Ly-6C (Gr-1) (BioLegend Cat# 108406) diluted in PBS at a 1: 1,000. For compensation, single stained samples were set. Cells were analyzed on BD FACSymphony (BD). Data were generated using FlowJo V10 (Treestar, Stanford, CA).

The immune cells in the BALFs of each mouse was directly counted using a hemocytometer, and then collected after the centrifugation at 716 (×g) for 5 min. The cells were resuspended and reacted with primary antibodies, including APC/Cy7 anti-mouse CD45 (biolegend, San Diego, CA), APC anti-human/mouse CD11b (biolegend, San Diego, CA), FITC anti-mouse Ly 6G/Ly 6C (Gr-1) (biolegend, San Diego, CA), PE anti-mouse F4/80 (biolegend, San Diego, CA), PE anti-mouse CD3ε (biolegend, San Diego, CA), FITC anti-mouse CD4 (bio legend, San Diego, CA) and APC anti-mouse CD8a (biolegend, San Diego, CA). After a 15-min incubation, the cells were washed with PBS twice, fixed with 2% paraformaldehyde, and then detected using flow cytometry under a BD FACSCanto system (Becton Dickinson FACS Calibur, Franklin Lakes, NJ, USA). 100,000 live-cell events per sample were analyzed by BD FACS Canto Clinical and FACS Diva software (Becton Dickinson, Franklin Lakes, NJ, USA), in which the Gr-1⁺/CD11b⁺/F4/80⁺ cells gated in Q2 were macrophages/monocytes, the Gr-1⁺/CD11b⁺/F4/80⁻ cells gated Q2-1 from CD45⁺ cells were neutrophils, and the CD45⁺/CD3⁺ cells gated from P1 were lymphocytes.