Pecy7 α cd45

For analyzing lung-infiltrating myeloid cells, PBS-perfused lung tissue (left lobe) was treated with a collagenase-D- and DNAse-1-containing tissue digestion buffer. Isolated cells were surface-immunolabeled for inflammatory monocytes (CD45⁺ CD11b⁺ Ly6C^hi) and neutrophils (CD45⁺ CD11b⁺ Ly6G^hi) and analyzed by FACS Aria-II flow cytometry. Briefly, ~5 × 10⁵–1 × 10⁶ lung cells were incubated with an Fc block (anti-CD16/32) for 10 min on ice. Following Fc blocking, the cells were washed with the FACS buffer (1× PBS, 2% FBS, and 0.05% sodium azide). Cell surface staining was then performed by labeling total lung cells with the following fluorochrome-conjugated monoclonal antibodies: PECy7 α-CD45 (clone: 30-F11); BV421 α-CD11b (clone: M1/70); PerCp-Cy5.5 α-Ly6C (clone: HK1.4); and FITC α-Ly6G (clone: 1A8) (all from BioLegend, San Diego, CA, USA, unless otherwise stated). Details of the cell surface immuno-labeling protocol for flow cytometry were described by Channappanavar et.al., 2020 [77 ]. A final concentration of 1:200 (antibody/FACS buffer) was used for all fluorochrome-conjugated antibodies, except for PerCp-Cy5.5-labeled antibodies used at a 1:300 concentration.