Differentiation of 3T3-L1 Pre-Adipocytes

Briefly, 3T3-L1 pre-adipocytes cell line (60159, Bioresource Collection and Research Center, Taiwan) was seeded to a 12-well culture plate with a cell density of 1 × 10⁴ per well and cultured at 37°C under 5% CO₂ in Dulbecco Modified Eagle Medium (DMEM, high glucose, 12800-017, Gibco, United States) supplemented with 10% calf bovine serum (16170-078, Gibco, United States) and 1% of 100X Anti-anti. After confluence, it were further cultured in starvation condition for 2 days to keep cells in the status of G₀/G₁ phase at least 85% in all population (Cao et al., 2012 (link)). The confluent 3T3-L1 cells were cultured in an adipo-differentiated medium to convert cells into adipocytes; where the adipo-differentiated medium was DMEM supplemented with 10% FBS, 1% of 100X Anti-anti, 1 mM dexamethasone (D4902, Sigma, United States), 0.2 M indomethacin (I7378, Sigma, United States), 0.1% insulin and 0.25 M 3-Isobutyl-1-methylxanthine (IBMX, I5879, Sigma, United States). The adipocytes were cultured in DMEM supplemented 10% FBS and 1% of 100X Anti-anti; and medium was refreshed every 3 days, until the oil droplets were observed by a fluorescence microscope (TS-100, Nikon, Japan) stained with Nile red (N1142, Invitrogen, United States) (Park et al., 2017 (link)).