Im currently differentiating 3T3-L1 cells in 96 well. After changing to differentiation media for 72 hours, I replace with insulin media, but the lipids droplets that formed disappears. until maintanance media, there are no lipid droplets observed. Can anybody help me how to troubleshoot this? Ive tried so many times now
Hello Dayana Cheong
Usually, lipid droplets should appear within 4-7 days after differentiation is induced. They will look extremely small initially. Lipid accumulation continues throughout the first two weeks. The lipid droplets gradually fuse to several big locules.
You mentioned that the lipids droplets formed disappears, and that there are no lipid droplets observed. One possible reason may be that the preadipocytes may not be differentiating well. This could be because the cells may have been passaged too many times.
Please note that contact inhibited 3T3-L1 cells are adipogenic. 3T3-L1 cells can lose contact inhibition after several weeks if they are densely cultured. So, loss of contact inhibition will result in loss of adipogenic potential. Thus, the differentiation potential of 3T3-L1 is dependent on contact inhibition. I feel you should use cells of a lower passage number (P6 - P10).
Also, ensure that cells are 100% confluent and that you wait two days post confluence before changing to differentiation media. The two days post-confluence will cause the cells to ball-up and start looking a little ugly, ready to be in a state to differentiate in the differentiation media.
Best.
Hello Malcolm Nobre
I need your advice, please
I seeded 3T3-L1 preadipocytes into well plates. there was a problem when refreshing the culture medium with FBS medium before initiating differentiation. When I added the FBS medium, the cells in all wells were detached and peeled off. I very gently replace the culture medium.
Passage 5-8. I seed 5,000 cells for 96 wells and 60,000 cells for 12 wells. I've had this problem before.
The agents I use have not changed. I had this problem only in some passage cells. I wonder what it might be about. I subcultured the cells at 70% without confluence.
Malcolm Nobre
thank you for your advice
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