I am trying to differentiate 3T3L1 cells into adipocytes, any helpful suggestions?
We tried to establish the differentiated 3T3-L1 cells as cell culture model in our lab. In the beginning we wanted to find out an advanced method to characterize the differentiation status of our cells over a series of experiments. Staining with Oil Red O alone did not satisfied us. But until today we have no good alternative.
But we learnt many things about the ticks of these cells to get reproducible results. First, the undifferentiated cells should never (!) come to 100% confluence before splitting. Second, the two days of post confluence are very important for the differentiation. Third, in our opinion the cells still have some rests of primary cell character. Next, the cells do not well differentiate in 96 well plates compared to 24 well plates, it seems the more surface the merrier.
In literature, you can find many differentiation protocols. It seems like everybody has his own protocol. We have good experiences with the protocol from ZenBio
Sometimes, some GH can be added with the differentiation medium.
Materials
Dulbecco's Modified Eagle's Medium, Ham's nutirient F-12, and antibiotic-antimycotic will be purchased from Invitrogen (Carlsbad, CA). Calf serum (CS), and trypsin-EDTA solution will be purchased from ATCC. Bovine transferrin, bovine insulin, bovine fetuin, mouse epidermal growth factor (EGF), 3,3’,5-Triidothyronine (T3), Dexamethasone, and isobutylmethylxanthine will be purchased from Sigma (St. Louis, MO). [3H] 2-deoxyglucose will be obtained from Perkin Elmer (Boston, MA). All the cell culture plastic wares will be purchased from VWR (West Chester, PA).
Cell Culture
Murine 3T3-L1 cells will be maintained as described (7,9). Briefly, stock cells will be maintained in DMEM supplemented with 10% calf serum (CS medium) in a humidified atmosphere of 5% CO2 without reaching confluence. For differentiation assays, cells will be disaggregated by 0.125% trypsin with 1 mM EDTA. Cell will be re-plated at a density of 6 x102 cells/cm2 in 6-wells culture plates in DMEM (10% CS) supplemented with antibiotics. At confluence, the medium will be replaced by the differentiation medium (DM) consisting of a basal medium (Ham's F-12:DMEM, 2:1), bovine fetuin (50um/ml), EGF (50ng/ml), T3 (100pg/ml), and transferrin (10ug/ml), supplemented with dexamethasone (1uM), 3-isobutyl-methylxanthine (0.8 mM), and insulin (5ug/ml), in a serum free culture.
Hello, I found these cells very easy to use in adipogenic studies.
Murine preadipocyte cell line 3T3-L1 was grown in DMEM medium, supplemented with New Born Calf Serum (NBCS) at 37°C in a humidified atmosphere of 5% CO2. Regarding the differentiation process, you can find useful information in the following paper: "Modulation of Adipogenic Conditions for Prospective Use of
hADSCs in Adipose Tissue Engineering" Int. J. Mol. Sci. 2012, 13, 15881-15900; doi:10.3390/ijms131215881 (in this paper I published my results on hADSCs, but 3T3-L1 work similarly).
Good luck !
We tried to establish the differentiated 3T3-L1 cells as cell culture model in our lab. In the beginning we wanted to find out an advanced method to characterize the differentiation status of our cells over a series of experiments. Staining with Oil Red O alone did not satisfied us. But until today we have no good alternative.
But we learnt many things about the ticks of these cells to get reproducible results. First, the undifferentiated cells should never (!) come to 100% confluence before splitting. Second, the two days of post confluence are very important for the differentiation. Third, in our opinion the cells still have some rests of primary cell character. Next, the cells do not well differentiate in 96 well plates compared to 24 well plates, it seems the more surface the merrier.
In literature, you can find many differentiation protocols. It seems like everybody has his own protocol. We have good experiences with the protocol from ZenBio
I always try to differentiate some neuroblastoma cell line to different cell types. For example, I employed RA for neuron differentiation, and BrdU for Glial or Swanna cell type, Hopefully it's helpful for your exmperiment.
Thanks for detailing your experienc Jannette. Can yuu share the link for the protocol.
Jannette, I might suggest using GPDH assays as a marker of differentiation. We have used GPDH Activity Measurement Kit (Takara, Japan) .
I added the file with the protocol. Additional, because of the "light primary cell character" the cells should be used only till passage 13. And, we find out that it is also important to use NCS (newborn calf serum) instead of FCS for the preadipocyte cultivation.
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