Cd45 af647

Dissected adipose tissue was cut into small pieces and suspended in RPMI (Sigma, USA) with 20% heat-inactivated FCS (Lonza, Switzerland) before homogenization for 1 hour in a shaker-incubator at 37 °C with 2 mg/mL collagenase II (C6885, Sigma, USA). Homogenized adipose tissue was processed as previously described^{55 (link)} using FACS buffer (PBS with 1% heat-inactivated FCS and 0.1% NaN₃) without fixation and permeabilisation. Cells were recorded on a LSRII (BD Biosciences, USA) flow cytometer, and data further analysed using Flowjo software (V10.0.7, Treestar). Gating strategies are shown in Fig. S6. The following antibodies for surface staining were used: CD45/PerCP (BioLegend, 30-F11), Siglec-F/PE (BD, E50-2440), CD11b/V500 (BD, M1/70), F4/80/APC (BioLegend, BM8), CD11c/APC-Cy7 (BD, HL3), CD45/AF647 (BD, 30-F11), CD4/AF488 (eBioscience, GK1.5), CD8a/FITC (BD, 53–6.7), CD11b/FITC (eBioscience, M1/70), CD49b/FITC (eBioscience, HMa2), F4/80/FITC (eBioscience, BM8), NK1.1/FITC (BD, PK136), FcεR1/PerCP-eF710 (eBioscience, MAR-1), CD19/PerCP-Cy5.5 (eBioscience, 1D3), CD11c/PE-cy7 (BD, HL3), ST2-biotin (MD biosciences, 101001B), Streptavidin-PE (eBioscience).

BASCs and type II alveolar (AT2) cells were isolated from pups on PND14 (n = 4) as described previously [16 (link)]. Briefly, lung tissues were digested and a single cell suspension was obtained before staining and cell sorting using a BD Influx system (BD Biosciences). The cells were stained using the following antibody panel: CD45-AF647, CD31-FITC, EpCAM-PE, Sca1 (Ly-6A/E)-BV421 from BD Biosciences with propidium iodide (PI) for exclusion of dead cells. BASCs were gated as the CD45^negCD31^negSca-1^posEpCAM^pos population, and AT2 cells were gated as the CD45^negCD31^negSca-1^negEpCAM^pos population (Fig. 8a).
Freshly isolated AT2 cells and BASC cells (2000 cells per well) from each experimental group were mixed with Matrigel containing mouse endothelial cells (20,000 cells per well). Cells were co-cultured and plated in triplicate using transwell inserts (6.5 mm, pore size 5.0 μm). Images were taken after 2 weeks in culture, and colonies picked after 3 weeks in culture for immunohistochemical analysis. Cultured AT2 cell and BASC colonies were fixed with 4% (w/v) paraformaldehyde for 30 minutes at room temperature and then immobilised with HistoGel (Thermo Scientific, MA, USA) for paraffin embedding. Colony sections were then deparaffinised in xylene and rehydrated before staining for BASC and AT2 cells.