2.10. Adipocyte cell culture

3T3-L1 cells were purchased from ATCC and cultured in DMEM supplemented with 10% FBS, 1 mM sodium pyruvate, and 1% Pen/Strep. The cells were tested for mycoplasma every three months. To differentiate the 3T3-L1 cells to adipocytes, the cells were induced with growth media supplemented with 1 μM dexamethasone, 0.5 mM IBMX, and 1 μg/mL insulin for 48 h and then maintained in growth media supplemented with only 1 μg/mL insulin. Stable doxycycline-inducible 3T3-L1 cells were generated by transducing the cells with lentivirus at an MOI ∼100, followed by selection with 1.5 μg/mL puromycin. Conditioned media were collected in OptiMEM I reduced serum media.