Percp cy5.5 conjugated anti f4 80

Lung digests were obtained by incubation with 1 mg/mL collagenase A, and 100 ng/mL DNase (Sigma) for 1 hour. 0.5‐1 × 10(6) cells from lung digests and BAL were stained with cell surface antibodies including PerCP‐cy5.5‐conjugated anti‐F4/80, PE‐Cy7‐conjugated anti‐Ly6G, PE‐conjugated anti‐CD45, BV421‐conjugated anti‐Siglec F and APC‐conjugated anti‐CD206 (BioLegend). Analysis was performed on FACScan cytometer (Becton Dickinson). All data were analysed on FlowJo software (Tree Star).

Isolated liver leukocytes or PBMCs were blocked with 5 μg/mL anti-mouse CD16/CD32 antibodies (BD Bioscience) and stained with fluorochrome-conjugated antibodies for 30 min at 4 °C in the dark. The cells were washed twice with PBS containing 3% FBS and fixed in 0.5 mL of Fluorofix buffer (BioLegend) for 30 min. The fixed cells were washed with PBS containing 3% FBS once and then analyzed on a FACSFortessa instrument (BD Bioscience). The fluorochrome-conjugated antibodies used in this study included FITC-conjugated anti-CD3, FITC-conjugated anti-CD11b, phycoerythrin-conjugated anti-CD45, APC-conjugated anti-CD8, APC-conjugated anti-CD115, APC/Cy7-conjugated anti-CD45, APC/Cy7-conjugated anti-Ly6G, BV605-conjugated anti-CD4, BV605-conjugated anti-Ly6G, BV421-conjugated anti-Ly6C, PerCP/Cy5.5-conjugated anti-CD19, PerCP/Cy5.5-conjugated anti-F4/80 (all purchased from BioLegend) and BV421-conjugated anti-CD11a (BD Bioscience). The compensation settings of the flow cytometer were determined with single-stained Ultracomp eBeads (Thermo Fisher Scientific). The frequency of each cell subpopulation obtained by flow cytometric analysis was further multiplied by the total isolated cell count to obtain the cell number for each subpopulation.