Cell Culture

1. Culture 3T3-L1 cells in 10% calf media (DMEM; high glucose, containing 10% BCSS and 1X PSG; see Note 4) at 37°C and 8% CO2 on 10-cm culture dishes (see Note 5)

2. Once plated, change the 10% calf media every 4 d and allow the fibroblasts (preadipocytes) to grow at least 2 d past confluence (approx 8 d postplating).

3. At 2 d past confluence, the cells can be differentiated by removing the 10% calf media and adding differentiation media (DMEM; high glucose, containing 10% FBS, 1X PSG, DMX, 10 iL/100 mL; insulin, 100 iL/100mL; and IBMX, 200 lL/100mL).

4. At 4 d after initiation of differentiation (approx 12 d postplating), the differentiation media is removed and an insulin media (DMEM; high glucose, containing 10% FBS, 1X PSG, and insulin, 100 iL/100 mL) is added.

5. At 4 d after initiation of insulin media (approx 16 d postplating), the insulin media is removed and growth media (DMEM; high glucose, containing 10% FBS and 1X PSG) is added. At this point, the cells can be used over the next 4 d (see Note 6).

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