Live dead eflour506

BAL cells were blocked with 1% BSA containing TruStain fcX (anti-mouse CD16/32) antibody (101319; BioLegend), followed by staining with antibodies. Antibodies used: Rat anti-mouse CD45-PE (12–0451–82; eBiosciences), LIVE Dead-eflour506 (65–0866; Invitrogen), Rat anti-mouse CD11b-APC-Cy7 (101225; BioLegend), anti-mouse CD64-PE-Cy7 (139313; BioLegend), Rat anti-mouse Ly6G-AF700 (561236; BD), Rat anti-mouse Siglec F-APC (155507; BioLegend), and Rat anti-mouse Ly6C: eflour450 (48–5932–82; Invitrogen). Hierarchical gating strategy was used to represent the resident alveolar macrophages as CD45⁺CD11b^+/−Ly6G⁻CD64⁺Ly6c⁻Siglec F^hi and monocyte-derived macrophages as CD45⁺CD11b^+/−Ly6G⁻CD64⁺Ly6c⁻Siglec F^low. To determine cell number, CountBright Absolute Counting Beads (Molecular Probes) were added according to manufacturer's instructions. Briefly, counting beads were gently vortexed and 50 μl was added to stained cells in a volume of 300 μl. Counting beads were gated on forward versus linear side scatter. The following equation was used to determine cell number: (number of cell events/number of bead events) × (lot specific bead count per 50 μl/volume of sample). Data was acquired on FACSAria II or LSR II (BD Biosciences) using BD FACS DIVA software (version 8.0.1). Data was analyzed using FlowJo (FlowJo LLC) software (Version 10.5.0).

Macrophages were stained with MitoTracker green (50 nM) for mitochondrial localization. Data were collected with a Becton Dickinson LSR‐II flow cytometer using FACS Diva software (BD Biosciences) and further analyzed with FlowJo software (version 8.5.2; TreeStar). BAL cells were blocked with 1% BSA containing TruStain fcX (anti‐mouse CD16/32) antibody (101319; BioLegend), followed by staining with antibodies. Antibodies used: Rat anti‐mouse CD45‐PE (12‐0451‐82; eBiosciences), LIVE Dead‐eflour506 (65‐0866; Invitrogen), Rat anti‐mouse CD11b‐APC‐Cy7 (101225; BioLegend), anti‐mouse CD64‐PE‐Cy7 (139313; BioLegend), Rat anti‐mouse Ly6G‐AF700 (561236; BD), Rat anti‐mouse Siglec F‐APC (155507; BioLegend), Rat anti‐mouse Ly6C: eflour450 (48‐5932‐82; Invitrogen), Rat anti‐mouse MHC II‐PerCP‐Cy5.5 (562363; BD), and Dead Cell Apoptosis Kit with Annexin V FITC and PI (V13242, Molecular Probes). Hierarchical gating strategy was used to represent the resident alveolar macrophages as CD45⁺CD11b^+/−Ly6G⁻CD64⁺Ly6c⁻Siglec F^hi and monocyte‐derived macrophages as CD45⁺CD11b^+/−Ly6G⁻CD64⁺Ly6c⁻Siglec F^low. Data were acquired on FACSAria II or LSR II (BD Biosciences) using BD FACS Diva software (version 8.0.1). Data were analyzed using FlowJo (FlowJo LLC) software (Version 10.5.0).