Insulin

The frozen 3T3-L1 adipose precursor cells (Shanghai Bio Leaf Biotech Co., Shanghai, China) were resuscitated, passaged, and plated. Take a 6-well plate as an example, Transfection is performed when the cell density reaches about 75%. About 6 h before transfection, we changed the medium to a serum-free medium without dual antibody. We added 2 μg of recombinant plasmid and 2 μL of transfection reagent to the anti-serum-free medium, vortex to mix, and let stand for 20 min. We added the mixed solution to a petri dish, put it in the incubator for 6 h, and then replaced it with normal culture medium. After 48 h, we added induction solution I (IBMX: 0.5 mmol/L, TOPSCIENCE, Shanghai, China; Dex: 1 μmol/L, Solarbio, Beijing, China; insulin: 10 μg/mL, TOPSCIENCE, Shanghai, China) for two days, then added induction solution II (insulin: 10 μg/mL) for 8 days. Every two days, we changed the induction liquid II once. After 8 days, the cells were collected for further processing.

The cryopreserved 3T3-L1 adipocytes were recovered, passaged and planked. Taking the 6-well plate as an example, when the cell density reached about 90%, after contact inhibition for 2 days, inducible solution I (IBMX: 0.5 mmol/L, TOPSCIENCE, Shanghai, China; Dex: 1 μmol/L, Solarbio, Beijing, China; insulin: 10 μg/mL, TOPSCIENCE, Shanghai, China) was added to the culture for 2 days, then inducible solution II (insulin: 10ug/mL) was added to the culture for 8–10 days, and inducible solution II was changed every 2 days. After that, the medium was replaced with 10% serum double antibody-free medium, and the cells were transfected after 24 h of culture. We added 2 μg of recombinant plasmid and 2 μL of lipo2000 (Invitrogen, Waltham, MA, USA) to the Opti (Thermo Fisher, Shanghai, China), mixed them by vortexing, and let the solution stand for 20 min. We added the mixed solution to a Petri dish, put it in the incubator for 6 h and then replaced it with normal culture medium. After 24 or 48 h, cells were collected for further processing.