Anti st2

The suspended SVFs from adipose depots of 10-wk-old male mice were fixed, blocked, and stained with conjugated antibodies, including anti-CD45, anti-Siglec-5, anti-CD11b, and anti-CD206 (eBioscience and BioLegend), to identify macrophage subsets. To detect ILC2s (CD45⁺Lin⁻CD90.2⁺ST2⁺), SVF cells were incubated with conjugated anti-CD45, anti-Lin (CD3e, CD11b, B220, CD11c, and Gr-1), anti-CD90.2, and anti-ST2 (eBioscience and BioLegend) after fixation unless specified otherwise. In Fig. S1 D, we used additional markers, including anti-RORC and anti-GATA3 (eBioscience), to gate ILC2s (CD45⁺Lin⁻CD90.2⁺RORC⁻ST2⁺GATA3⁺). Anti-AMPK α 1 (phospho-T183) and AMPK α 2 (phospho-T172) antibody (ab23875; Abcam), eFluor 660–conjugated anti-phospho-IκBα (S32/S36; eBioscience), and PE-conjugated phospho-IKKα/β (Ser176/180; Cell Signaling) were used to detect phospho-AMPK, phospho-IκBα and phospho-IKK in ILC2. For the staining of GATA3, RORC and phosphorylated proteins, cells were permeabilized with 0.25% Triton X-100 for 20 min after fixation. FACS analysis was performed on a FACS Calibur (BD PharMingen), and the data were analyzed with FlowJo software as described previously (Dong et al., 2013 (link)). The gating strategy used for ILC2s, eosinophils, and macrophages in adipose tissue is shown in Fig. S3, I–K.

Lymphocytes were isolated from lung and tumor tissue by digestion with collagenase A (1 mg/ml; Roche) and DNase I (0.5 µg/ml; Roche) in isolation buffer (RPMI 1640 supplemented with 5% FBS, 1% l-glutamine, 1% penicillin-streptomycin, and 10 mM Hepes) for 30 min at 37°C. Cells were filtered through 100-µm cell strainers, washed in isolation buffer, and stained in PBS supplemented with 0.25% BSA, 2 mM EDTA, and 0.1% sodium azide. Antibodies used included anti-CD45, anti-Foxp3, anti–IL-18Rα, anti-ST2, anti-CD62L, anti-CD103, anti–PD-1, anti-GITR, anti–CTLA-4, anti-KLRG1, anti-Ki67, anti-CD5, anti-NK1.1, anti-CD45R (B220), anti–IL-17, anti–IL-4, anti-IFNγ, and anti–Ly-6C (eBioscience); anti-TCRβ, anti-CD3, anti-CD4, anti-CD8, anti-CD127, anti-CD11B, anti-MHCII, and anti-Gr1 (BioLegend); anti-CD44 and anti-CD25 (Tonbo); anti–IL-5 and anti-TNFα (BD PharMingen); and anti-AREG (R&D Systems). For exclusion of dead cells, samples were first stained with Ghost Dye (Tonbo) cell viability reagent. Intracellular staining for cytokines, Foxp3, AREG, Ki-67, and CTLA-4 was performed using the Foxp3/transcription factor staining buffer set (eBioscience) as per manufacturer’s protocol. The H2-K^b OVA_257–264 tetramer was obtained from the NIH Tetramer Core Facility.