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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

1

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. 
2

To DMEM, add IBMX to a final concentration of 0.5 mM (1 mL IBMX stock solution per 100 mL medium)

3

Add dexamethasone to a final concentration of 1 µM (100 µL dexamethasone stock solution per 100 mL medium)

4

Add insulin to a final concentration of 10 µg/mL

Stage 2 - Differentiation of 3T3-L1 cells into adipocyte-like cells

Steps

1

Seed cells

Seed cells in a six-well plate at a density of 3x103 cells per cm2.

2

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.

3

To initiate differentiation, remove DMEM and add 2–3 mL MDI induction medium per well (Day 0).

4

On Day 3, remove MDI induction medium from the cells and replace with 2–3 mL insulin medium.

5

On Day 6, remove insulin medium from the cells and add fresh DMEM.

6

By Day 7–10, fully differentiated adipocyte-like cells should be obtained.