Multicolor Flow Cytometry Analysis of Alveolar Macrophages

BAL cells were incubated for 10 min at 4 °C with an anti-mouse CD16/32 antibody (1:200; Biolegend, San Diego, CA) to block non-specific binding, followed by incubation with a FITC-conjugated anti-mouse CD11b (1:200; Biolegend, San Diego, CA) and an AF 700-conjugated anti-mouse CD11c (1:200, Biolegend) antibodies for 30 min at 4 °C. Data were collected on a BD LSR Fortessa flow cytometer using FACSDiva software (Becton Dickinson, Mountain View, CA) and analyzed using FlowJo software (Tree Star, San Carlos, CA). A range of 20,000–50,000 cells was analyzed per sample. The initial gating eliminated debris and red blood cells. Next, consecutive gates, were set to identify CD11c⁺ and CD11b⁺ cells, as previously described (Swirski et al. 2009 (link); Valdés-Ferrer et al. 2013 (link)). The relative expression of CD11c was used to distinguish CD11c-positive alveolar macrophages (CD11c⁺, CD11b⁺) and CD11c-negative infiltrated cells (CD11c^neg, CD11b⁺). Fluorescence minus one (FMO) controls were used for proper gating.