A Chemically-induced Method of Adipogenesis

Technical Document

At least two different distribution lots of 3T3-L1 Mouse Embryonic Fibroblasts were tested for their ability to differentiate in response to a mixture of insulin, dexamethasone, and methylisobutylxanthine (IBMX). The assay was established according to published protocols and is relatively easy to perform. The components listed here represent reagents that can be obtained individually from commercial suppliers.

Supplies available from ATCC

3T3-L1 Mouse Embryonic Fibroblasts, ATCC CL-173
Dulbecco’s Modified Eagle’s Medium (DMEM), ATCC 30-2002
Bovine Calf Serum, ATCC 30-2030
Fetal Bovine Serum (FBS), ATCC 30-2020

Additional supplies

Insulin (bovine), Sigma Aldrich I0516
Dexamethasone, G Biosciences API-04
IBMX (Sigma Aldrich I5879)

Media formulations used throughout

Medium
Pre-adipocyte Expansion Medium	90% DMEM 10% Bovine Calf Serum
Differentiation Medium	90% DMEM 10% FBS 1.0 µM Dexamethasone 0.5 mM IBMX 1.0 µg/mL Insulin
Adipocyte Maintenance Medium	90% DMEM 10% FBS 1.0 µg/mL Insulin
Pre-adipocyte Expansion Procedure	Maintain the cells in the Pre-adipocyte Expansion Medium according to instructions for handling provided in the 3T3-L1 product sheet. Seed the cells at about 3000 cells per cm² and never allow the cells to become confluent. Subculture 3T3-L1 cells before cultures reach 70% confluence (Figure 1).

Figure 1. Light microscopy of subconfluent 3T3-L1 cells.

Differentiation procedure

When the cells are 70-80% confluent, harvest the cells by trypsinization according to the instructions related to subculture.
Seed the cells in the desired culture vessel in the Pre-adipocyte Expansion Medium.

Culture vessel	Cell density
6 well plate	8 × 10⁴ cells/well
24 well plate	2 × 10⁴ cells/well
96 well plate	2 × 10³ cells/well

Let the cells grow for 48 hours, or until the culture reaches 100% confluence (Figure 2 & Figure 3). Feed the cells with the Pre-adipocyte Expansion Medium after 48 hours.

Figure 2. Light microscopy of confluent 3T3-L1 cells.

Figure 3. Confluent 3T3-L1 cells stained with Nile Red prior to induction.

Incubate the cells as a confluent culture for another 48 hours.
Remove the growth medium from each well and add an identical volume of Differentiation Medium.
Incubate in Differentiation Medium for 48 hours.
After 48 hours, replace the Differentiation Medium with Adipocyte Maintenance Medium.
NOTE: At this stage, the cells detach easily from the tissue culture vessel. Gentle pipetting/handling is recommended from this point forward.
Replace the Adipocyte Maintenance Medium every 48 to 72 hours.
The cells should be fully differentiated between 7 to 15 days after induction, as evidenced by observation of lipid droplet formation (Figure 4 & Figure 5).

Figure 4. Light microscopy of differentiated 3T3-L1 cells.

Figure 5. Differentiated 3T3-L1 cells stained with Nile Red.

Why do the cells peel off the substrate when I add the Differentiation Medium?

Possible cause: pH and osmolality of the Differentiation Medium fall outside of the normal physiological range.

Solution: Check pH and osmolality of the Differentiation Medium prior to use. pH should fall between 7.0 and 7.4, and osmolality should falls within 270 to 365 mOsm/kg.

Possible cause: Cells were left for too long without a liquid layer.

Solution: Take great care to immediately add Differentiation Medium to the well once the Pre-adipocyte Expansion Medium is removed.

Why do the cells detach after I add the Adipocyte Maintenance Medium?

Possible cause: The differentiated cells were handled too harshly and detached.

Solution: The cells easily detach once differentiation occurs. Take care when adding or removing medium. If the cells detach, add fresh medium to spent cultures and incubate for 48 hours.

Possible cause: Cells were left for too long without a liquid layer. pH and osmolality of the Adipocyte Maintenance Medium fall outside of the normal physiological range.

Solution: Check pH and osmolality of the Adipocyte Maintenance Medium prior to use. pH should fall between 7.0 and 7.4, and osmolality should fall within 270 to 365 mOsm/kg.

Possible cause: Cells were left for too long without a liquid layer.

Solution: Take great care to immediately add Adipocyte Maintenance Medium to the well once the Differentiation Medium is removed.

Why haven’t the cells formed lipid droplets after 2 weeks?

Possible cause: The wrong reagents were used (eg human insulin was substituted for bovine insulin).

Solution: If using an alternative supplier, be sure to check the quality, purity and/or characterization (eg cell-culture-tested) of the material prior to use.

Possible cause: The differentiation reagents were added to the medium in the wrong amounts.

Solution: Over- or under-supplementing the Differentiation Medium could keep the cells from forming lipid droplets; it may also, in the case of over-supplementing, cause irreversible harm to the cells.

Possible cause: The cells were not confluent prior to the addition of the Differentiation Medium.

Solution: Be sure to allow the culture to reach 100% confluence prior to changing to the Differentiation Medium (ie at confluence, the cells are already primed to differentiate).

Possible cause: The cells may not have been exposed to the Differentiation Medium long enough.

Solution: Once the Differentiation Medium is added, be sure to expose the cells for a full 48 hours.

Possible cause: Passage effect.

Solution: The cells may have been over-passaged or kept too dense during routine subculture.

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Products used in this technical document

3T3-L1 Mouse Embryonic Fibroblasts

These cells can undergo a pre-adipose to adipose-like conversion as they progress from a mitotic to a contact-inhibited state.

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Fetal Bovine Serum

A high-quality serum tested and confirmed to support the culture and cryopreservation of many different cell lines.

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Gloved hands holding flask containing Dulbecco's Modified Eagle Medium (DMEM).

Dulbecco’s Modified Eagle’s Medium (DMEM)

This extensively used basal medium can be used to support the growth of a wide variety of human and animal cell lines.

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A herd of about 20 cows walking in the same direction in a field with blue skies overhead.

Calf Bovine Serum

Iron-fortified serum able to support the growth of cells using sequential growth curves.

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