Pe percp cy5.5 α ly6c

For phenotypic analyses of lung infiltrating immune cells, lungs collected at different days post-infection, PBS perfused lungs (left lobe were cut into small pieces, treated with collagenase-D and DNAse1 for 30 minutes at room temperature, followed by homogenization of lung pieces using a 3ml syringe plunger flang/thumb rest. Homogenized cells were passed through 70µM strainer to obtain single cell suspension. Isolated single cell suspension was surface immunolabelled for neutrophil (CD45⁺ CD11b⁺ Ly6G^hi) and inflammatory monocyte (CD45⁺ CD11b⁺ Ly6c^hi) markers by flow cytometry. For cell surface staining, lung cells were labelled with the following fluorochrome-conjugated monoclonal antibodies: PECy7 α-CD45 (clone: 30-F11); FITC α-Ly6G (clone: 1A8); PE/PerCp-Cy5.5 α-Ly6C (clone: HK1.4); V450 α-CD11b (clone: M1/70); APC α-F4/80 (clone: BM8) (all procured from Biolegend). A detailed cell surface and intracellular immunolabelling protocol for flow cytometry studies are described in our recent publication (61 (link)). All fluorochrome-conjugated antibodies were used at a final concentration of 1:200 (antibody: FACS buffer), except for FITC labeled antibodies used at 1:100 concentration.

For phenotypic analyses of lung infiltrating immune cells, PBS-perfused lungs were treated with collagenase-D and DNAse1 for 30 min at room temperature, followed by homogenization of lung pieces using a 3 mL syringe plunger flang/thumb rest. Isolated single-cell suspension was surface immunolabeled for neutrophil (CD45⁺ CD11b⁺ Ly6G^hi) and inflammatory monocyte (CD45⁺ CD11b⁺ Ly6c^hi) markers by flow cytometry. For cell surface staining, lung cells were labeled with the following fluorochrome-conjugated monoclonal antibodies: PECy7 α-CD45 (clone: 30-F11); FITC α-Ly6G (clone: 1A8); PE/PerCp-Cy5.5 α-Ly6C (clone: HK1.4); V450 α-CD11b (clone: M1/70); APC α-F4/80 (clone: BM8) (all procured from BioLegend). A detailed cell surface and intracellular immunolabeling protocol for flow cytometry studies is described in our recent publication (70 (link)). All fluorochrome-conjugated antibodies were used at a final concentration of 1:200 (antibody: FACS buffer), except for FITC-labeled antibodies used at 1:100 concentration.