Hey,
so within the last week I tried to differentiate wildtype MEF's into adipocytes using adipogenic medium (ADM) containing Insulin, Dexamethason, Indomeathcin, Isobutyxanthin. After 8 days of ADM treatment there were only few to none MEF's differentiated. The one's that did I tried to stain with Oil Red O. I prepared fresh Stock solution (3mg in 100ml Isoprop), diluted it freshly with water (2:3) and filtered it. However, even after the third time I did the staining (including different incubation times) none of the adipocytes became red. We now had the Idea that what we assumed to be adipocytes is really something else. Do you have any suggestions? I'll attach a picture of it.
Thanks a lot!
You will need to optimize the differentiation condition probably. As these are primary cells, it takes longer time to differentiate. Kindly optimize adipogenic differentiation. The lipid droplets are visible in some of the cells but very less %tage.
I totally agree with the others. Differentiation protocols might be adjusted for your cells. It is not unusual that differentiation starts at an later time point than anticipated when you use primary cells. And Oil red o staining only helps you to better visualize the droplets within the cells. But usually you can already see the droplets in the light microscope when screening your cultures without staining the cells.
Thanks a lot for your answers!
I used Insulin at a concentration of 1 µg/µl, IBMX 500 µM, Indomethacin 50 µM and Dexamethason 1 µM. Our protocol worked in previous experiments actually pretty well. We thawed up new cells today to check if it is a problem with the cells. What is the function of Rosiglitazone?
@Anil & Arvind: Do you think what we can see in the pictures are really lipid droplets? Or is it possible that this is something else?
The phase contrast images indicate that the adipogenesis was not success because I could not clearly see the lipid drop in the culture. I have two suggestion:
1. Extend the differentiation duration up to 3 weeks.
2. You can use adipogenesis kit from invitrogen.
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