Differentiation of 3T3-L1 cells into adipocyte-like cells: case study

The mouse embryonic fibroblast cell line 3T3-L1 can be chemically induced to differentiate into adipocyte-like cells. 3T3-L1 differentiation is an economical and convenient way to generate adipocyte-like cells for experiments.

In this case study, we tested adipocyte characteristics such as lipid accumulation, production of leptin and adipocyte marker expression as 3T3-L1 cells are chemically induced to go down the adipocyte line.

3T3-L1 cell differentiation into adipose-like cells

To induce 3T3-L1 cells to differentiate into adipocyte-like cells, cells were treated with isobutylmethylxanthine (IBMX, ab120840), insulin (ab123768) and dexamethosone (ab120743).

Results

3T3-L1 cells were characterized for adipocyte traits before and after differentiation into adipocyte-like cells.

Oil Red O staining

During adipocyte differentiation, cells accumulate lipid. Oil Red O staining kit (ab150678) was used to visualize lipid in 3T3-L1 cells and adipocyte-like cells. As expected, lipid accumulates in 3T3-L1 cells after differentiation into adipocyte-like cells.

Figure 1. Oil Red O staining of 3T3-L1 and adipocyte-like cells. Cells were stained using our Oil Red O staining kit.

Detection of leptin using cell lysates

Leptin is a hormone that is primarily secreted by adipocytes and is involved in regulating hunger. Leptin mouse ELISA kit (ab100718) was used to test leptin levels in cell lysates of 3T3-L1 cells and adipocyte-like cells. As shown in Figure 2, leptin levels were increased in the cell lysates of adipocyte-like cells compared with 3T3-L1 cells.

Figure 2. Leptin assay. Mouse (Ms) leptin was quantified in lysates of adipocyte-like cells (3T3-L1 adip.lys.) and 3T3-L1 cells (3T3-L1 lys.). Leptin was detected by ELISA using our Leptin mouse ELISA kit.

Detection of adipocyte markers by immunofluorescence

To further test the chemically induced differentiation from 3T3-L1 to adipocyte-like cells, immunofluorescence was performed to detect the presence of adipocyte markers adiponectin and FABP4.

Proteins were detected using primary antibodies against target proteins and Alexa Fluor® conjugated secondary antibodies.

Figure 3. Detection of adiponectin by immunofluorescence. Adiponectin (green) was detected using adiponectin primary antibody (ab22554; 2.5 µl/mL). Beta tubulin (red) was detected using our rabbit polyclonal (ab6046) antibody. Cells were imaged by confocal microscopy, using z-stack for adipocyte-like cells.

Adiponectin is a protein produced by adipocytes (Scherer et al., 1995). It acts as a hormone and has an important role in modulating glucose and lipid metabolism in insulin sensitive tissues (Yamauchi et al., 2001).

Adiponectin was detected using our mouse monoclonal anti-adiponectin antibody (ab22554) and anti-mouse Alexa Fluor® secondary antibody (ab150120). The immunofluorescence images in Figure 3 show an increase in adiponectin in cells after chemically induced differentiation into adipocyte-like cells.

Figure 4. Detection of FABP4 by immunofluorescence. FABP4 (green) was detected using FABP4 primary antibody (ab92501; diluted 1/1000). Alpha tubulin (red) was detected using our mouse monoclonal (ab7291) antibody. Cells were imaged by confocal microscopy, using z-stack for adipocyte-like cells.

FABP4 is a fatty acid binding protein located in adipocytes (Baxa et al., 1989). FABP4 was detected using our anti-FABP4 rabbit monoclonal antibody (ab92501) and anti-rabbit Alexa Fluor® secondary antibody (ab150088).

Detection of FABP4 by immunofluorescence showed an absence of FABP4 before chemical induced differentiation. However, FABP4 was strongly detected in some cells after differentiation (Figure 4).

Glucose uptake by adipocyte-like cells

Increased glucose uptake after stimulation with insulin is a characteristic of adipose cells (Cushman & Wardzala, 1980). We used our glucose uptake assay kit (ab136955) to test glucose uptake in adipocytes before and after addition of insulin.

Glucose uptake is measured by providing 2-deoxyglucose (2-DG) to the cells. 2-DG is taken up by glucose transporters and metabolized to 2-DG 6-phosphate (2-DG6P), which is measued as it accumulates in cells. The data show an increase in glucose uptake of over five-fold in adipocytes after exposure to insulin.

Figure 5. Glucose uptake assay. Glucose (2-DG) uptake by adipocyte-like cells was measured in the presence and absence of insulin (I). Glucose uptake was assessed using our glucose uptake assay kit.

Conclusion

Differentiation of 3T3-L1 cells into adipocyte-like cells is accompanied by lipid accumulation, adiponectin production, expression of adipocyte markers and sensitivity of glucose uptake to insulin. Using a range of our kits and assays, we were able to sucessfully track the appearance of these characteristics in 3T3-L1 cells after differentiation.

References

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