Alexa fluor 700 conjugated anti mouse cd45

BAL cells were centrifuged (400 x g for 6 min) and resuspended in 700 μl of red blood cell (RBC) lysis buffer (Sigma Aldrich) for 5 min at room temperature. Cells were then centrifuged (400 x g, 6 min) and resuspended in 100 μl of staining buffer (PBS containing 2% FBS and 0.02% sodium azide). This was followed by incubation for 10 min at 4°C with anti-mouse CD16/32 (1:100; BioLegend, San Diego, CA) to block non-specific binding, and then with FITC-conjugated anti-mouse CD11b (1:200; BioLegend), PE-CF594-conjugated anti-mouse Ly6C (1:200; BD Biosciences, San Jose, CA), PerCP-Cy5.5-conjugated anti-mouse Siglec F (1:200; BD Biosciences), PECy7-conjugated anti-mouse F4/80 (1:200; BioLegend), AlexaFluor647-conjugated anti-mouse Ly6G (1:200; BioLegend), AlexaFluor700-conjugated anti-mouse CD45 (1:200; BioLegend), Brilliant violet 421-conjugated anti-mouse CD11c (1:200; BioLegend) antibodies or appropriate isotypic controls for 30 min, followed by eFluor780-conjugated fixable viability dye (eBioscience, San Diego, CA) for 30 min. Cells were washed twice with staining buffer, fixed with 3% paraformaldehyde, and analyzed on a Gallios flow cytometer (Beckman Coulter Inc., Brea, CA). Data were analyzed using Beckman Coulter Kaluza version 2.1 software. Viable cells (10,000) were assessed for expression of CD45, followed by CD11b, Ly6G, Ly6C, F4/80, CD11c, and Siglec F.