Fluorescence-activated Cell Sorting (FACS).

Adipocytes and the Stromal Vascular Fraction (SVF) were isolated from mouse BAT following the procedure described previously⁵. The interscapular BAT was dissected, finely minced, and digested for 45 minutes using a cocktail containing type 1 collagenase (1.5 mg ml−1; Worthington Biochemical), dispase II (2.5 U ml−1; Stemcell Technologies), and fatty acid-free BSA (2%; Gemini Bio-Products) in Hanks’ balanced salt solution (Corning Hanks’ Balanced Salt Solution, with calcium and magnesium). The resulting dissociated tissue was subsequently centrifuged at 500g and 4°C for 10 minutes. Adipocytes, located in the uppermost layer, were gently collected using a wide-mouthed transfer pipette and filtered through a 100 µm cell strainer. Brown adipocytes were allowed to float for 5 min at room temperature before they were centrifuged at 30g for 5 minutes at room temperature. This cycle was repeated three times, after which the adipocytes were immediately lysed in Trizol. For the SVF isolation, the pellet was resuspended in 10 ml of 10% FBS in DMEM, filtered through a 100 µm cell strainer into a fresh 50-ml tube, and subsequently centrifuged at 500g for 7 minutes. Red blood cells were lysed in 2 ml of sterile ammonium–chloride–potassium lysis buffer (ACK Lysing Buffer, Lonza) for 5 minutes on ice. The cells were then filtered once more through a 40-μm cell strainer, washed with 20 ml of a solution containing 10% FBS in DMEM, and centrifuged at 500g for 7 minutes. The cells were resuspended in 1 ml of Cell Staining Buffer (BioLegend) before proceeding with staining.

Cells were stained using the fluorescently conjugated antibodies as outlined in Supplementary Table 2. The cells were then incubated with the antibodies at the specified dilutions from Supplementary Table 2 for a duration of 30 minutes, followed by two rounds of washing in Cell Staining Buffer (BioLegend). Cells were sorted using an SH800 sorter using a 100 µm sorting chip (Sony Biotechnology). Debris and doublets were excluded based on forward and side scatter gating, and 7-AAD was used to exclude dead cells. Following sorting, cells were centrifuged at 300 g for 5 minutes and lysed in Trizol for subsequent RNA isolation and gene expression analysis.