Pdgfrα-positive cell isolation

To isolate PDGFRα⁺ cells from WATi, magnetic-activated cell sorting (MACS) were used. For the best yield of PDGFRα⁺ cells WATi of three 8-week old WT C57Bl/6J mice was pooled. Tissue was minced and digested in digestion buffer (DMEM containing 0.5% BSA and 1.5 mg ml⁻¹ Collagenase II). After digestion, all tissue debris was removed by filtration using a 100-µm nylon mesh (Merck Milipore, NY1H00010). Samples were centrifuged and the pellet was washed with 2 ml of ice-cold MACS buffer (0.5% BSA, 2 mM EDTA, 1% P/S in PBS pH 7.2). Cells were counted and FcR Blocking reagent and PDGFRα MicroBeads (Miltenyi Biotec, 130-101-502) were added to the cell suspension. After 15 min of incubation, cells were washed with MACS buffer, centrifuged and resuspended in MACS buffer. Liquid seperation columns (Miltenyi Biotec, 130-042-401) were rinsed with ice-cold MACS buffer and cell suspension was applied onto the column. The column was washed with MACS buffer and the flow through containing unlabelled cells was collected. PDGFRα⁺ cells were flushed out by pushing the plunger into the column after adding MACS buffer.