Single cell isolation from SVF

Single cell isolation from SVF- Inguinal white adipose tissue (iWAT) from mice treated with saline or CL were dissected and placed on sterile 6-well tissue culture plate with ice-cold 1X DPBS. Fat pads were blotted on a napkin to removed excess liquid. Tissues were cut and minced with scissors and placed in 15 ml conical tubes containing digestion buffer (2 ml DPBS and Collagenase II at 3 mg/ml; Worthington Biochemical, Lakewood, NJ, USA) and incubated at 37 °C for 40 min with gentle shaking at 100rpm. Following tissue digestion 8 ml of resuspension media (DMEM/F12 with glutamax supplemented with 15%FBS and 1% pen/strep; Thermo Scientific, CA) was added to stop enzyme activity. The digestion mixture was passed through 100 μm cell strainer and centrifuged at 150 x g for 8 min at room temperature. The pellet was resuspended and incubated in RBC lysis buffer (Thermo Scientific, CA) for 3 min at room temperature to remove red blood cells followed by centrifugation at 150 x g for 8 min. The pellet was resuspened in resuspension media and spun down again at 150 x g for 8 min.  Finally, the cell pellet was resuspended in 1 ml of 0.01% BSA (in DPBS). This final cell suspension solution was passed through a 40 μm cell strainer (Fisher Scientific, Hampton, NH, USA) to discard debris and cell number was counted for Drop-Seq application.