Hi Francisca,

Please see Rodrigues et al. Molecular and Cellular Endocrinology, 2012; 361(1-2):69-79.

Basically, cells should be grown until confluence and incubated, 2 days later, with DMEM containing 10% FBS, 10 ug/ml insulin, 250 nM dexamethasone and 0.5 mM IBMX. After 3 days, the medium should be replaced to DMEM, FBS and insulin (same concentrations) for further 3 days. At this time, cells should be already differentiated into adipocytes. You should also know that, when too old, 3T3-L1 cells loose their ability to differentiate. You need to check how many passages the cells have. With more than 20 it might be difficult to assure high levels of differentiation.

Hope this help.

Adriana

Hi Francisca,

Please see the following papers for detail.

http://www.ncbi.nlm.nih.gov/pubmed/25183280

http://www.sciencedirect.com/science/article/pii/S221052391300024X

Cell density, cell passage number, and the concentration of insulin, dexamethasone, and IBMX are very important for the differentiation of 3T3-L1 adipocytes. If you need further details, please let me know. 

Thanks,

Bhesh

Differentiation will be faster if you include a PPARg ligand such as Rosiglitazone with the induction cocktail.

The protocol I use is DMEM with 10% FBS (with PS) to culture and on confluence induce with 100nM insulin, 500uM IBMX, 250nM Dexamethasone and 2uM rosiglitazone for 48 hrs in DMEM+10% FBS (with PS) and then change the media to DMEM + 10% FBS(with PS) containing 100nM insulin alone for another 48 hrs. Again change the medium DMEM+10% FBS(with PS) until the cells are completely differentiated. Generally it takes two more changes. 

Thanks for all your responses,  Im new in this research, it's been six days since the differentiation and  I have observed very small granules.  Espero que resulte todo bien.

Your question is very good

Best Regards