Anti mouse cd16 32 antibodies

Mouse lung tissues were collected at experimental end point, digested with 1 mg/ml collagenase A (Roche) and 30 µg/ml DNase I (Sigma-Aldrich) in complete RPMI-1640 media for 30 min at 37°C, and mechanically minced. Digested tissues were then passed through a 70-µm strainer (Thermo Fisher Scientific) to single-cell suspension and treated with ACK Lysing Buffer (Thermo Fisher Scientific) to remove red blood cells. Cells were resuspended in Live/Dead Fixable Aqua (Thermo Fisher Scientific) for 20 min at 4°C. Following a wash, cells were blocked with anti-mouse CD16/32 antibodies (BioXCell) for 30 min at 4°C. Cocktails of staining antibodies were added directly to this mixture for 30 min at 4°C. Prior to analysis, mouse cells were washed and resuspended in 100 µl 4% PFA for 30–45 min at 4°C. Following this incubation, cells were washed and prepared for analysis on an Attune NXT (Thermo Fisher Scientific). Data were analyzed using FlowJo software version 10.6 software (Tree Star). The specific sets of markers used to identify each subset of cells are summarized in Fig. S5.