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3T3-L1 Differentiation Protocol

Materials
- Dulbeco’s Modified Eagles Medium (DMEM, GibcoBRL-Cat# 11965-084: high glucose, with L-glutamine, with pyroxidine HCl, without sodium pyruvate)
- Calf Serum (GibcoBRL-Cat# 10437-028/Lot # 1026566), filter sterilize (0.22 μL filter) before mixing into DMEM
- Isobutylmethylxanthine (IBMX; Sigma I-7018)
Make Fresh: Dissolve IBMX in 0.3 N KOH to a final concentration of 0.0115 g/mL (50mM stock), filter sterilize with a 0.22 mm syringe filter
- Dexamethasone (Sigma D-4902)
Stock: 10 mM in 100% Absolute Ethanol (add a drop of NaOH if not soluble). Dilute to 1 mM in PBS, filter sterilize with a 0.22 mm syringe filter
- Insulin (Bovine; Sigma I-5500)
Stock: 174.4uM (1 mg/mL) in 0.02 M HCl, filter sterilize with a 0.22 mm syringe filter
- MEM Sodium Pyruvate (100mM; GibcoBRL-Cat# 11360-070)
- Pen/Strep/Glutamine (100x P/S/G; GibcoBRL-Cat# 10378-016)

Solutions
- 8% Calf Serum/DMEM (Proliferation Media)
  • 40 mL Calf Serum
  • 6 mL MEM Sodium Pyruvate
  • 6 mL 100x P/S/G
  • 500 mL DMEM
- 10% FBS/DMEM (Differentiation Media)
  • 50 mL Fetal Bovine Serum
  • 6 mL 100 mM MEM Sodium Pyruvate
  • 6 mL 100x P/S/G
  • 500 mL DMEM
- MDI Induction Media (to required volume)
  • 1:100 IBMX (final 500uM)
  • 1:1,000 Insulin (final 1ug/mL)
  • 1:10,000 Dexamethasone (final 1uM)
  • In required volume of differentiation media (10% FBS/DMEM)
- Insulin Media (to required volume)
  • 1:1,000 Insulin (final 1ug/mL)
  • In required volume of differentiation media (10% FBS/DMEM)
Method
- Preadipocyte maintenance and passage:
Plate the cells in proliferation media (8% CS/DMEM) on treated polysterene culture dishes from Corning and incubate them at 37 ˚C in 10% CO2. It is important to feed the preadipocytes every couple of days to avoid letting them get too confluent (>70%), if you want to continue to passage them and differentiate them at a later date. Take care to split them appropriately. They can be split as far as 1:15, though it is usually done 1:10 or less depending on need.

- Adipocyte Differentiation Protocol
  1. Grow preadipocytes to confluency in proliferation media (8% calf serum/DMEM)
  2. Two days post-confluency (Day 0), stimulate the cells with MDI induction media. You will notice a distinct change in the morphology of the cells (become more spindly) in the next two days
  3. After MDI (Day 2), change the media to insulin media. The media will begin to get more viscous as free fatty acids are produced by the cells and secreted into the media.
  4. Two days later (Day 4), change the media to differentiation media (10% FBS/DMEM). Feed cells with differentiation media every two days. Full differentiation is usually achieved by day 8.