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.

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3T3-L1 differentiation is an economical and convenient way to generate adipocyte-like cells for experiments.

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.

Preparation of MDI induction medium

• Prepare stock solutions of IBMX (50 mM) and dexamethasone (1 mM) 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

Preparation of insulin medium

• To DMEM, add insulin to a final concentration of 10 µg/mL

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

1. 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. Conflency 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.

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.

Protocol adapted from Reed BC, Lane D (1980). Insulin receptor synthesis and turnover in differentiating 3T3-L1 preadipocytes. Proc. Natl. Acad. Sci. USA. 77, 285–289