Differentiation of 3T3-L1 fibroblasts to adipocytes

Tuesday, June 14, 2005

Description
We maintain the stock of 3T3-L1 cells in 150 mm tissue culture dishes in DMEM (Gibco catalog no. 11995-065) supplemented with 10% Fetal Bovine Serum, 100 units/ml of Penicillin and 100 礸/mL of Streptomycin. The cell culture is split 1:5 every 3-4 days when the cells are covering the plate but not packed tightly. In order to maintain rapid growth and potential to differentiate it is important not to let the cells overgrow!
The cells can be carried to at least passage 12 provided that the fibroblast are maintained properly.

Procedure
After 7-8 days in culture, the cells will be quiescent and we start the Differentiation Schedule by changing the media to DMEM supplemented as above plus 4 礸/mL of insulin (Sigma catalog no. I-5500), 115 礸/mL of 3-Isobutil 1-methyl xanthine (Sigma catalog no. I-5879) and 97.5 ng/mL of Dexamethasone (Sigma catalog no. D-1756). After 3 days, carefully remove the media and replace with DMEN plus serum and antibiotics. The cells are responsive as soon as they start to accumulate fat droplets (usually apparent at day 4).

Recipes

Supplies

Tips
It is important to note that special care must be given to the cells after they have been differentiated. The monolayer is very fragile within the first 5 days of differentiation and can be damaged easily by aspirating or addition of media.

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Differentiation of 3T3-L1 fibroblasts to adipocytes