Anti sca 1 microbeads

After obtaining the SVF, adipose progenitors were isolated as described previously (Bayindir et al, 2015; Babaei et al, 2017). In brief, the SVF cell pellet was resuspended in the appropriate volume of BSA buffer and preincubated with FcBlock (anti‐CD16/32; eBioscience, Frankfurt, Germany) for 10 minutes on ice. Cells were then stained with biotin‐conjugated lineage antibodies, namely Ter119 (TER‐119), CD31 (390), and CD45 (30‐F11) antibodies (eBioscience), for 30 min on ice to label erythrocytes, endothelial cells, and hematopoietic cells, respectively. After staining, cells were washed and incubated with Streptavidin MicroBeads (130‐048‐102, Miltenyi Biotec, Bergisch Gladbach, Germany) for magnetic separation with an OctoMACS Separator according to the manufacturer's instructions. Ter119⁻CD31^–CD45^– (Lin⁻) cells in the flow‐through were incubated with Anti‐Sca‐1 MicroBeads (130‐106‐641, Miltenyi Biotec) for magnetic separation following the manufacturer's protocol. The Lin⁻Sca1⁺ cells retained in the column were collected by removing the column from the magnet and plunging through with 1 ml BSA buffer. The cells were then centrifuged at 300 g for 5 min at 4°C for further processing.

Female C57BL6 mice were obtained from Janvier. EpSCs were isolated from telogen back skin collected from P56-60 mice, as previously described [27 (link)] with the following changes: fat and muscle tissue were removed from back skin using a scalpel. The skin was incubated in 0.5% Trypsin-EDTA (10X; Gibco,15400054) for 25 min at 37 °C on an orbital shaker. A single-cell suspension was then obtained by scraping the skin with a scalpel followed by neutralizing the trypsin by adding 1X PBS buffer containing 2% chelexed FBS (PBS-FBS(-)) (Gibco; 10010-015). The resulting cell suspension was then filtered through 70 µm and 40 μm cell strainers (Corning; 431750, 431751) and spun down. EpSCs were isolated using magnetic-associated cell sorting using Anti-SCA-1 microbeads (Miltenyi Biotec, 130-106-641) and a MACS MultiStand system (Miltenyi Biotec) together with MS columns (Miltenyi Biotec, 130-042-201). The resulting SCA-1+ EpSCs were spun down and resuspended in Trizol LS (Thermo Fisher, 102960-10). RNA was isolated using the Direct-zol™ RNA MiniPrep kit (Zymo Research, R2050) and concentration was determined using the Qubit^TM RNA BR assay kit (Invitrogen, Q10210). All animal procedures were approved by the Veterinary Office of the Canton of Zurich, Switzerland (License ZH233/2019).