Alexafluor647 anti cd301

Mouse lungs were dissociated into single-cell suspensions using enzymatic digestion with the Lung Dissociation Kit (Miltenyi Biotec 130-095-927) and a GentleMACS Dissociator (Miltenyi Biotec) following the manufacturer’s protocol. One million cells per sample were incubated in Live/Dead Fix Blue (ThermoFisher L23105) for 20 minutes at 4°C and then washed and incubated with Fc Block (anti-mouse CD16/32) (BD Pharminogen 553142) for 10 minutes at room temperature and appropriate antibodies were added for 30 minutes at 4°C (Pacific Orange anti-CD45 (Invitrogen MCD4530), PE anti-F4/80 (BioLegend 123109), APC-eFluor780 anti-CD11b (Invitrogen 47-0112-82), PE-Cy7 anti CD11c (BioLegend 117317), and AlexaFluor647 anti-CD301 (BioLegend 145603)). Cells were then washed twice, fixed with 1% paraformaldehyde, and analyzed with the BD LSRII flow cytometer and FlowJo v10.7 software. For characterization of M1- and M2-polarized macrophage populations, CD45⁺F4/80^highCD11b^high populations were distinguished based on surface expression of CD11c (M1 marker) or CD301 (M2 marker)^{19 (link)}. Resident and recruited macrophage populations were defined as F4/80⁺CD11b⁻CD11c⁺ and F4/80⁺CD11b⁺CD11c⁻ populations, respectively, as described previously^{20 (link)}.