2. Learning Centers
  3. Scientific Reviews
  4. Adipogenic Differentiation of 3T3-L1 Cells: A Complete Protocol & Product Guide
Adipogenic Differentiation of 3T3-L1 Cells: A Complete Protocol & Product Guide

Obesity is characterized by an increase in the mass or dysfunction of adipose tissue resulting from an imbalance between energy intake and expenditure. It is closely associated with metabolic disorders such as diabetes, cardiovascular diseases, non-alcoholic fatty liver disease, and certain types of cancer[1]. The expansion of fat depots—particularly white adipose tissue—is marked by either an increase in adipocyte size (hypertrophy) or the formation of new adipocytes from precursor cells (hyperplasia)[2].Therefore, inducing adipogenic differentiation in cells plays a crucial role in the study of obesity-related disease mechanisms and the development of therapeutic drugs.

1. Methods for Adipogenic Induction

3T3-L1 cells are fibroblasts isolated from mouse embryos. Due to their ability to differentiate from fibroblasts into adipocytes, they are widely used in studies of adipogenesis and the biochemistry of adipocytes[3][4].

Figure 1: Morphology of 3T3-L1 Cells[1].

To induce the transformation of 3T3-L1 cells from a fibroblast phenotype into adipocytes, the earliest method used was the “cocktail method.” This involves treating growth-arrested cells with adipogenic inducers, most commonly insulin, dexamethasone, and 3-isobutyl-1-methylxanthine (IBMX), at typical concentrations of 1µg/ml, 0.25µM, and 0.5mM, respectively[5][6][7].About four days after the addition of these reagents, the cells begin to accumulate lipids in the form of lipid droplets. Over time, both the number and size of the lipid droplets increase.

However, this method has relatively low differentiation efficiency, and its effectiveness decreases rapidly with increasing passage number—particularly when using cells that have been stored in liquid nitrogen. To address this issue, Katja Zebisch and colleagues found that adding rosiglitazone, a PPARγ agonist, to the standard cocktail significantly improves differentiation efficiency. This modified protocol has since become a widely used and classic method for inducing adipogenesis in 3T3-L1 cells[4].

Figure 2: The Effect of Rosiglitazone on 3T3-L1 Cell Differentiation[4].

Microscopy Images: 3T3-L1 preadipocytes (control) and 3T3-L1 cells undergoing adipocyte differentiation for 14 days at passage 6 and passage 10, with or without rosiglitazone treatment. Images of stained cell culture dishes were captured using a digital camera. Individual 3T3-L1 cells were photographed using a 25× inverted microscope.(B) Schematic diagram of the 3T3-L1 differentiation process in differentiation media with or without rosiglitazone.

2、Preparation of Adipogenic Induction Medium

Prepare the following stock solutions according to the formulation in Table 1. Aliquot and store at -80°C.

Table 1: Formulation of Adipogenic Induction Medium Stock Solution

Additionally, MCE provides a water-soluble form of dexamethasone, HY-14648C, with a solubility in water of ≥ 100 mg/mL, suitable for induction experiments with different protocols.

Preparation of Working Solutions (using 50 mL of culture medium as an example)

2.1. Adipogenic Induction Differentiation Medium A components:

Add 50 µL of 1 mM Dexamethasone (DXM), 50 µL of 500 mM IBMX, 50 µL of 2 mM Rosiglitazone, and 50 µL of 10 mg/mL insulin stock solution into 50 mL of high-glucose DMEM medium containing 10% FBS and 1% PBS.

2.2. Adipogenic Induction Differentiation Medium B components:

Add 50 µL of 10 mg/mL insulin stock solution into high-glucose DMEM medium containing 10% FBS and 1% PBS.

3. Adipogenic Induction Culture

Seed logarithmic-phase 3T3-L1 cells into culture vessels at an appropriate density. Incubate at 37 °C in a 5% CO₂ atmosphere until the cells reach 70–80% confluency.

Remove the culture medium and add Adipogenic Induction Medium A. After 2 days of induction, remove Medium A and replace it with Adipogenic Induction Medium B. After maintaining the cells in Medium B for 2 days, remove it and switch back to Medium A. Alternate between Medium A and B, observing cell morphology daily.

Repeat the induction and maintenance cycles until a sufficient number of lipid droplets with appropriate size appear. Once this is achieved, proceed to staining.

Figure 3: Adipogenic Induction Results by an MCE Customer[8].

A.3T3-L1 induction group; B. Dedifferentiation of 3T3-L1 adipocytes induced by hyperosmotic conditions (MCE products used: HY-P0035, HY-14648, and HY-12318).

4. Analysis of Adipogenic Induction

4.1 Qualitative Analysis:

Staining methods for intracellular lipid droplets (LDs) in 3T3-L1 cells mainly include Oil Red O staining, Nile Red staining, and BODIPY lipid droplet dyes.

4.2 Semi-Quantitative Analysis:

Dissolve 0.35 g of Oil Red O in 100 mL of pure isopropanol. Fix 3T3-L1 cells with 3.7% formaldehyde at room temperature for 1 hour, then wash twice and dry completely using a hairdryer. Stain the fixed cells with Oil Red O diluted with distilled water (6:4) at room temperature for 1 hour. Wash the cells four times with distilled water. Extract the Oil Red O stain using 100–200 µL of isopropanol, and measure the absorbance at 490 nm[9][10].

Figure 4: 3T3-L1 Induction Staining and Semi-Quantitative Results[10].

3T3-L1 cells were induced to differentiate after overexpressing Stomatin tagged with RFP (hSTOM-RFP), using RFP alone as the control. Lipid content in the resulting adipocyte-like cells was assessed by staining and semi-quantitative analysis.

4.3 Measurement of Lipid Droplet Size

Two small lipid droplets (LDs) can fuse to form a larger LD. Larger LDs may result from the gradual accumulation of lipids from smaller LD vesicles, filling into a single larger LD. Therefore, measuring the size of individual LDs is essential for studying LD fusion. After staining, images of adipogenic differentiation can be captured and analyzed using image analysis software (such as ImageScope) to assess the size of adipocytes and the area of individual lipid droplets[10].

Figure 5: STOM Knockdown Cells Exhibit More Small LDs and Fewer Large LDs[10].

3T3-L1 cells were stained with BODIPY-FL and observed under Differential Interference Contrast (DIC) and fluorescence (Flu) microscopy. Lipid droplet size was then measured using ImageScope software.

5. Notes for Adipogenic Induction

a. When adding inducers to the cells, handle gently. Use a pipette tip to slowly add the solution along the wall of the culture vessel to minimize disturbance. Otherwise, cells may detach or curl up. Alternatively, pre-coating the culture plate with 0.1% gelatin before seeding can help improve cell adherence.

b. The passage number of the cells directly affects the induction efficiency. Higher passage numbers lead to weaker adipogenic potential and require a longer induction period. In such cases, the concentrations of dexamethasone and insulin can be increased appropriately.

c. From the start of induction to the end of differentiation, typically two rounds of induction are needed. Lipid droplets usually begin to appear around day 6, with the following 2 days allowing for lipid accumulation. If differentiation is efficient, lipid droplets may appear as early as day 4.

d. Whenever possible, prepare working solutions fresh before use.

Product Recommendation

Dexamethasone

Glucocorticoid receptor agonist.

IBMX

Broad-spectrum phosphodiesterase inhibitor.

Rosiglitazone

Orally active selective PPARγ agonist.

Insulin (human)

Regulate glucose levels

Penicillin-Streptomycin (100×), Sterile

Dual antibiotics, sterile filtered, ready for direct use in cell culture.

DMEM (High Glucose, L-Glutamine, Pyruvate, Phenol Red, no HEPES)

Suitable for culturing various mammalian cells, as well as primary fibroblasts, neurons, glial cells, human umbilical vein endothelial cells, smooth muscle cells, and others.

BODIPY-FL

Used to investigate sphingolipid internalization, transport, and endocytosis in mouse embryonic stem cells.
References
[1] Schwartz MW, et al. Obesity Pathogenesis: An Endocrine Society Scientific Statement. Endocr Rev. 2017;38(4):267-296.
[2] Jo J, et al. Hypertrophy and/or Hyperplasia: Dynamics of Adipose Tissue Growth. PLoS Comput Biol. 2009;5(3):e1000324.
[3] Howard Green, et al. Sublines of mouse 3T3 cells that accumulate lipid. Cell. 1974 March:Volume 1, Issue 3p 113-116.
[4] Poulos SP, et al. Cell line models for differentiation: preadipocytes and adipocytes. Exp Biol Med (Maywood). 2010;235(10):1185-1193.
[5] Green H, et al. An established pre-adipose cell line and its differentiation in culture. Cell. 1974 Oct;3(2):127-33.
[6] Rubin CS, et al. Development of hormone receptors and hormonal responsiveness in vitro. Insulin receptors and insulin sensitivity in the preadipocyte and adipocyte forms of 3T3-L1 cells. J Biol Chem. 1978 Oct 25;253(20):7570-8.
[7] Russell TR, et al. Conversion of 3T3 fibroblasts into adipose cells: triggering of differentiation by prostaglandin F2alpha and 1-methyl-3-isobutyl xanthine. Proc Natl Acad Sci U S A. 1976 Dec;73(12):4516-20.
[8] Liu G, et al. Hypertonicity induces mitochondrial extracellular vesicles (MEVs) that activate TNF-α and β-catenin signaling to promote adipocyte dedifferentiation. Stem Cell Res Ther. 2023;14(1):333.
[9] Green H, et al. An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion. Cell. 1975;5(1):19-27.
[10] Wu SC, et al. Stomatin modulates adipogenesis through the ERK pathway and regulates fatty acid uptake and lipid droplet growth. Nat Commun. 2022;13(1):4174. Published 2022 Jul 19.