3T3-L1 differentiation is an economical and convenient way to generate adipocyte-like cells for experiments.
Preparation of media must be carried out in a tissue culture hood under aseptic conditions. MDI (methylisobutylxanthine, dexamethasone, insulin) induction medium and insulin medium should be freshly prepared.
Prepare stock solutions.
To DMEM, add IBMX to a final concentration of 0.5 mM (1 mL IBMX stock solution per 100 mL medium)
Add dexamethasone to a final concentration of 1 µM (100 µL dexamethasone stock solution per 100 mL medium)
Add insulin to a final concentration of 10 µg/mL
Seed cells
Seed cells in a six-well plate at a density of 3x103 cells per cm2.
Grow cells in DMEM until a confluency of 70% is reached, changing the medium every 2–3 days.
Confluencey should not exceed 70% before differentiation as this increases cell death after differentiation.
To initiate differentiation, remove DMEM and add 2–3 mL MDI induction medium per well (Day 0).
On Day 3, remove MDI induction medium from the cells and replace with 2–3 mL insulin medium.
On Day 6, remove insulin medium from the cells and add fresh DMEM.
By Day 7–10, fully differentiated adipocyte-like cells should be obtained.
Differentiation into adipocyte-like cells can be tracked by Oil Red O staining to monitor lipid accumulation, or by monitoring the expression of adipocyte markers such as adiponectin and FABP4.