7 aad solution

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.

After treatment, the BMDMs were incubated with 20 μM BrdU (B5002, Sigma) in fresh culture medium at 37 °C in a humidified atmosphere (5% CO₂). After 2 h, the cells were washed twice with PBS (without Ca²⁺/Mg²⁺) and harvested. Subsequently, 1 × 10⁶ cells were resuspended in cold (–20 °C) ethanol and fixed overnight at 4 °C. Afterward, the cells were washed and incubated in 2 M HCL containing 0.5% Triton X-100 at RT. After 30 min, the cells were washed again and incubated with 0.1 M Na2B4O7 for 10 min at RT. After another wash, the cell pellets were resuspended in anti-BrdU antibody solution (1:25, 3D4; BioLegend) and incubated for 30 min in the dark at RT. The cells were washed twice in cell staining buffer (BioLegend; 420201) and resuspended in 100 μL staining buffer containing 5 μL 7-AAD solution (BioLegend; 420403). After a 30 min incubation at RT in the dark, the cells were analyzed using a FACSAria II instrument (BD Biosciences).

Arachidonyl-2′-chloroethylamide hydrate (ACEA; CB1 agonist), Rimonabant hydrochloride (SR141716A; CB1 antagonist/inverse agonist), GW833972A (CB2 agonist), SR144528 (CB2 antagonist/inverse agonist), and dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) were used. These cannabinoid receptor agonists and antagonists were dissolved in DMSO at a concentration of 20 mM and stored at −20 °C until used.
Saponin (Amresco, Solon, OH, USA) was used. Carboxyfluorescein succinimidyl ester (CFSE), brefeldin A, and monensin were obtained from Sigma-Aldrich. RPMI 1640 medium and fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA) were used. Ficoll-Hypaque solution (IsoPrep)(Robbins Scientific Corporation, Sunnyvale, CA, USA) was used. Finally, 7-AAD solution was purchased from BioLegend.