GSM5707532: Lin- SVF cells from 2.5-month-old mice: sample2; Mus musculus; RNA-Seq
Mouse epididymal adipose tissue (eWAT) was minced and transferred to a 50 mL Falcon tube containing 10 mL digestion buffer (100mM HEPES pH 7.4; 120mM NaCl; 50mM KCl; 5mM Glucose; 1mM CaCl2, 1.5% BSA, and 1 mg/mL collagenase D). The tissue was incubated in a 37℃ shaking water bath for 30 min. Digestion was stopped by adding 35 mL 2% FBS in PBS. The solution was passed through 100 µm and then 40 µm strainers, and centrifuged at 400 g for 5 min. The supernatant was aspirated, and SVF pellet was collected. Lin- SVFs was obtained by Multicolor Flow Cytometry (MACS) (Stemcell Technologies, Cat# 17656). To obtain Lin- SVFs, anti-mouse-CD45-PE, anti-mouse-CD31-PE, and anti-mouse-Ter119-PE (1:200, BioLegend) were used.  To obtain immune cells from eWAT, only anti-mouse-CD45-PE was used and isolated by MACS. Human peripancreatic white adipose tissue was minced and transferred to a 50 mL Falcon tube containing 10 mL digestion buffer (1 × PBS, and 10 mg/mL collagenase D). The tissue was incubated in a 37℃ shaking water bath for 45 min. Digestion was stopped by adding 35 mL 2% FBS in PBS. The solution was passed through a 250 µm mesh (Spectrum), and then 100 µm and 40 µm strainers, and centrifuged at 600 g for 5 min. The supernatant was aspirated, and SVF pellet was collected. To obtain Lin- SVF, cells were incubated with anti-human-CD45-PE, anti-human-CD31-PE, and anti-human-CD235a-PE (1:200, BioLegend), and isolated by MACS (Stemcell Technologies, Cat# 17654). To obtain immune cells, only anti-human-CD45-PE (1:200, BioLegend) was used and isolated by MACS.