Differentiation of 3t3-L1 cells into adipocyte-like cells
This protocol outlines how to chemically induce the differentiation of 3T3-L1 cells into adipocyte-like cells, adapted from Reed & Lane (1980).
Last edited Mon 29 Aug 2022
3T3-L1 differentiation is an economical and convenient way to generate adipocyte-like cells for experiments.
Stage 1 - Preparation of media
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.
Steps
Prepare stock solutions.
- Prepare stock solutions of IBMX (50mM) and dexamethasone (1mM) in DMSO. If using lyophilized insulin, reconstitute this according to the manufacturer's instructions.
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.
Stage 2 - Differentiation of 3T3-L1 cells into adipocyte-like cells
Steps
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.