Mouse primary white preadipocytes (mWA) were isolated from the stromal vascular fraction (SVF) of the inguinal WAT of 4- to 6-week-old male C57BL/6 J mice (purchased from Model Animal Research Center of Nanjing University, China). All animal studies were approved by the Ethics Committees at the Nan Jing Medical University. The detached tissues were cut into small pieces in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA) containing 5% FBS (Gibco) and 0.2% collagenase I (Sigma-Aldrich, St. Louis, MO). After 60 mins of digestion at 37 °C, the digestion buffer was filtered through a 100 μm nylon mesh (Thermo Fisher Scientific, Waltham, MA, USA) and centrifuged at 1800 rpm for 10 mins. The pellet was then resuspended in DMEM growth medium (GM), supplemented with 10% FBS (Gibco) and 1% P/S (Gibco), and seeded onto a 6-well plate.
After two more days of 100% cell confluence, the culture medium was replaced by preadipocyte differentiation medium (PADM; ScienCell), containing 5% FBS (ScienCell), 1% preadipocyte differentiation supplement (PAdDS; ScienCell) and 1% P/S (ScienCell). Seven days after differentiation, the medium was changed to GM as previously described. The cells were then maintained in GM for 7 days when the lipid droplets could be sufficiently displayed. During the whole process of culture and differentiation, the medium was changed every 2 days.
After two more days of 100% cell confluence, the culture medium was replaced by preadipocyte differentiation medium (PADM; ScienCell), containing 5% FBS (ScienCell), 1% preadipocyte differentiation supplement (PAdDS; ScienCell) and 1% P/S (ScienCell). Seven days after differentiation, the medium was changed to GM as previously described. The cells were then maintained in GM for 7 days when the lipid droplets could be sufficiently displayed. During the whole process of culture and differentiation, the medium was changed every 2 days.