Anti cd16 32 2.4g2

2 × 10⁶ liver cells, or 20 μl of whole blood was incubated with Zombie Aqua fixable viability dye (Biolegend, London, UK) for 10 min at RT and then with 0.025 μg anti‐CD16/32 (2.4G2; Biolegend) in 10% normal mouse serum (Life Technologies, Paisley, UK). Cells were then incubated with antibodies (Supplemental Table 1). Cells were washed, spun at 300 g for 5 min and, where necessary, incubated with fluorescently labeled streptavidin. 7‐AAD solution (Biolegend) was added to samples 10 min before acquisition when comparing isolation protocols. DAPI was used as a viability marker for FACS. Liver cells were gated as shown, whereas alveolar and interstitial mϕ were identified as CD45⁺CD11c⁺SiglecF⁺ and CD45⁺CD11c⁺SiglecF⁻MHCII⁺CD64⁺ cells, respectively.
For BrdU and Ki67 staining, cells were fixed and permeabilized overnight in FoxP3/Transcription Factor Staining Buffer (eBioscience). Cells were washed in PermWash (eBioscience) and stained with anti‐Ki67 and anti‐BrdU antibodies.
Cells were acquired on a LSRFortessa (BD Biosciences, Wokingham, UK) or FACSAriaII (BD) at the QMRI Flow Cytometry and Cell Sorting Facility, University of Edinburgh, and data analyzed in FlowJo software (Tree Star, Ashland, Oregon). Fluorescence‐minus‐one controls were used to set gates.

Mouse CD1d tetramers coupled to APC and loaded with PBS-57 (an iNKT cell ligand analog to α-GalCer) were provided by the NIH Tetramer Core Facility. Fluorochrome-labeled monoclonal antibodies (mAbs) specific to mouse CD45.1 (A20), CD45.2 (104), TCRβ (H57-597), CD4 (RM4-5), NK1.1 (PK136), Gr-1 (RB6-8C5), CD11b (M1/70), CD69 (H1.2F3), IFN-γ (XMG1.2), IL-4 (11B11), and IL-17A (TC11-18H10.1) were all purchased from BioLegend. c-Maf mAb (sym0F1) was purchased from eBioscience. For intracellular cytokine staining, cells were incubated with PMA (50 ng/ml), ionomycin (500 ng/ml), and brefeldin A (10 µg/ml; Sigma) for 3 h. After preincubation with unconjugated anti-CD16/32 (2.4G2; BioLegend) mAb, cells were stained for surface antigen and then fixed and permeabilized using the IC fixation buffer and permeabilization buffer (eBioscience) followed by incubation with anti-cytokine or isotype control Abs. FACS data were acquired by BD LSRFortessa (BD Biosciences) and analyzed with FlowJo software. For cell sorting, murine naive CD4⁺CD62L^hi T cells and CD1d/PBS-57⁺TCRβ⁺ iNKT cells were sorted by FACSAria IIIu (BD Biosciences).

For flow cytometry analysis, murine cells were preincubated with anti-CD16/32 (2.4G2) to block Fc receptors and then surface stained for 20–30 min at 4°C with antibodies to CD3e (145-2C11), CD11b (M1/70), CD19 (1D3 or 6D5), CD27 (LG.7F9 or LG.3A10), CD45.1 (A20), CD45.2 (104), CD94 (18D3), CD122 (TM-b1), CD132 (TUGm2), CD49a (HM⍺1), DX5/CD49b (DX5), Ncr1 (29A1.4), NK1.1 (PK136), KLRG1 (2F1), Ly49D (4 × 10⁵), Ly49G2 (4D11), Ly49I (YLI-90), and Ly49H (3D10) (purchased from BioLegend or eBioscience).
Human cells were surface stained for 25 min at 4°C with antibodies anti-CD3 (Clone UCHT1; eBioscience) and anti-CD56 (Clone 5.1H11; BioLegend).
Streptavidin-conjugated fluorophores were purchased from BioLegend or eBioscience. Stainings were performed with appropriate combinations of fluorophores. Propidium iodide (0.05 μg/ml from Immunochemistry) was used as a live–dead marker. Events were collected on a FACS Canto, Fortessa (Becton Dickinson) and data were analyzed using FlowJo software (Tree Star Inc.).