Mouse adipose-derived stem cells (ASCs) were isolated from inguinal subcutaneous fat of C57BL/6 mice that were fed normal chow. The cells were cultured as described previously [12 (link)]. Briefly, subcutaneous adipose tissues were digested with collagenase type 1 (Sigma-Aldrich, St. Louis, MO, USA) in PBS (phosphate-buffered saline) by incubation in a shaker at 37 °C for 30 min. The digestion was terminated by the addition of 10% fetal bovine serum (FBS), and centrifuged at 1200 rpm for 5 min. Cells were suspended in complete medium made of DMEM/F12 (Dulbecco modified Eagle medium) medium supplemented with 10% FBS and 1% penicillin and streptomycin. Cells were cultured in 37 °C at 5% CO2 incubator. ASCs at passage 3–5 were used in all the experiments.
ASCs were identified by FACS as described previously [12 (link)] with fluorescence-conjugated anti-mouse antibodies, FITC Hamster Anti-Rat CD29 (BD Pharmingen; Cat.555005), Alexa Fluor 647 Rat anti-Mouse CD34 Clone RAM34 (RUO) (BD Pharmingen; Cat.560233), APC Rat Anti-Mouse CD90.2 Clone 53-2.1 (RUO) (BD Pharmingen; Cat.561974), APC anti-mouse CD105 (Biolegend; Cat.120413), PE anti-mouse CD31 (Biolegend; Cat.102407), or PE/Cy7 anti-mouse CD45 (Biolegend; Cat.103113), according to the manufacturer’s instructions. Flow cytometry was conducted on a Becton-Dickinson LSR I analyzer.
ASCs were identified by FACS as described previously [12 (link)] with fluorescence-conjugated anti-mouse antibodies, FITC Hamster Anti-Rat CD29 (BD Pharmingen; Cat.555005), Alexa Fluor 647 Rat anti-Mouse CD34 Clone RAM34 (RUO) (BD Pharmingen; Cat.560233), APC Rat Anti-Mouse CD90.2 Clone 53-2.1 (RUO) (BD Pharmingen; Cat.561974), APC anti-mouse CD105 (Biolegend; Cat.120413), PE anti-mouse CD31 (Biolegend; Cat.102407), or PE/Cy7 anti-mouse CD45 (Biolegend; Cat.103113), according to the manufacturer’s instructions. Flow cytometry was conducted on a Becton-Dickinson LSR I analyzer.