Differentiation of TL Fibroblasts

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1. Replace culture medium A with culture medium B and incubate for 2 d in the incubator.

2. After 2 d, replace culture medium B with freshly prepared differentiation medium (see Note 7) containing freshly prepared solutions of insulin, DMX, and IBMX (see Note 8)—10 mL for a 10-cm plate and 2.0 mL for a 35-mm plate.

3. After 48 h, the differentiation media is replaced with culture medium B containing 670 nM insulin alone (for 100 mL of culture medium B, add 100 iL of 4 mg/mL insulin; sterile filter) (see Note 9).

4. After 2 d and thereafter, add fresh culture medium B every 2 d (see Notes 5 and 9)

5. For best results, use the differentiated adipocytes for experiments between d 9 and 15 after initiation of differentiation. Adipocytes, after d 16 of post-differentiation initiation, start to die, lose their phenotype and become increasingly resistant to manipulations such as infection with adenoviruses or electroporation with DNA.

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