Pe cy7 conjugated ly6c clone hk1

BALF samples were washed with FACS buffer (10% BSA in PBS). Red blood cells were lysed using ammonium chloride and 10 cells/well were seeded into a 96-well U-bottom plate. Cells were pre-incubated with anti-FcgRIII/II (Fc block) in FACS buffer before a 30-min incubation with the following fluorochrome-labeled antibodies (Biolegend): PE-Cy7-conjugated Ly6C (clone HK1.4, dilution 1:200), PE-conjugated CD11b (clone M1/70, dilution 1:200), APC-conjugated Ly6G (clone 1A8, dilution 1:200), and FITC-conjugated CD11c (clone N418, dilution 1:100). Cells were washed with phosphate-buffered saline twice and counterstained with 7AAD (Biolegend) to differentiate apoptotic and dead cells and then analyzed using a LSR Fortessa (Becton Dickinson).

BALF samples were washed with FACS (fluorescence-activated cell sorting) buffer (2% BSA in PBS), followed by red blood cell lysis using Red Cell Lysis Buffer (Tiangen, Beijing, China). For the surface staining, cells were stained with the following fluorochrome-labeled antibodies (Biolegend, San Diego, USA): PE-Cy7-conjugated Ly6C (clone HK1.4, dilution 1:200), PE-conjugated CD11b (clone M1/70, dilution 1: 200), APC-conjugated Ly6G (clone 1A8, dilution 1: 200), and FITC-conjugated CD11c (clone N418, dilution 1: 100). Cells were washed with PBS twice and counterstained with 7AAD (Biolegend, San Diego, USA) to differentiate apoptotic and dead cells, then analyzed using a BD FACSVerse Cell Analyzer (BD, New Jersey, USA).