# Seed MEFs at 100% confluence in 12-well plates and grow for 2 days (D0).
# On day 0 (D0) treat MEFs with differentiation-inducing media containing insulin (830 nM), dexamethasone (1 µM), and 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM).
3. # At 48 hours, replace the media with the maintenance DMEM medium containing 10% FBS and insulin (830-nM), followed by a replenishment of the media every 2 days until the end of adipogenic differentiation (prior to cells lifting off).• '''Note1 :''' Cells should shrink after day 1, and then start to round up. By *day 3 cells should begin to accumulate lipid (the media should change color as well)4. # Protein lysate and RNA extraction is taken at day 4 (D3). • '''Note2: ''' In the 12-well plate, Tsc2-/- MEFs started to be detached from the plate on D3, and consequently lysates were taken at that point to ensure adequate sample collection.• '''Note3: ''' A 6-well plate can also be used to increase surface area and protein/RNA content from each treatment. Seeding density of 90% followed by 2 days of growth prior to adipogenic differentiation may increase adherence and maintenance of Tsc2-/- MEFs.
