I'm currently using this protocol for 3T3-L1 adipogenic differentiation:
- Seeding density: 10,000 cells/well (6-well plate). After cells grow to 100% confluency, they're left for another 48 hours post-confluent before differentiation.
- Cells then are fed with differentiation medium (Day 0): DMEM + 10% FBS + 1% Pen/Strep + 0.5mM IBMX + 1μM Dexamethasone + 10μg/mL Insulin.
- After 48 hours of induction (Day 2), the medium is switched to DMEM + 10% FBS + 1% Pen/Strep + 10μg/mL Insulin. Cell are then fed with this medium every 2 days. I usually do the Oil red staining on day 12-14.
Although I couldn't get a high percentage of adipocytes (usually ~20%) using this protocol, but I found that it's 4-5 times better than the protocol suggested by ATCC (please find it in the attachment below), which starts with 80,000 cells/well (6- well plate) and only 1μg/mL Insulin plus other components (IBMX, DEX) with the same concentration described above. When I used the protocol by ATCC, only
Hi,
You can see better adiposity using differentiation medium-1 0.5mM IBMX, 0.25uM Dexamethasone and 1ug/ml Insulin. And the differentiation medium-2 with 1ug/ml Insulin. I have obtained good results with high lipid accumulation. Try checking your incubation time you provide for the cells in differentiation medium. Also, make sure that your cells are post confluent.Because, 3T3-L1 needs 3 days to attain post confluency.
Refer these papers!
Hi Snehaa Chandrakumar,
Thanks for your suggestion and those helpful papers. I've also read some articles using those concentrations of inducing agents as you mentioned, so I'll try it next time and I hope the problems would be solved. I just wonder what the initial seeding density in your protocol is and how much lipid accumulation that you obtained from it (in percentage). Did you strictly follow the protocol provided in the first paper (Trigonelline attenuates the adipocyte differentiation and lipid accumulation in 3T3-L1 cells)?
Again, thank you in advance.
Yes I followed the same protocol and obtained good results. Good luck!
Dear Mai,
If your goal is to have high numbers of differentiated adipocytes, try to include a PPAR-gamma agonist during the first 2-3 days of differentiation, try e.g. rosiglitazone.
Thank you so much for your suggestion Johan. And I wonder if it works with 10T1/2 cells, as I also use this cell line for adipocyte differentiation. I couldn't find many effective protocols for the adipogenic differentiation of 10T1/2.
Dear Mai, I have no experience with 10T1/2 cells, but you can certainly try the PPAR gamma agonist with these cells as well.
Thank you again Johan. By the way, what is the concentration of rosiglitazone that you recommend, because I haven't used this agent before. If possible, could you please suggest some reliable papers for reference?
Dear May, we used PPAR gamma agonists in concentrations of 7-10 uM. You can check the paper of Bouwman et al. Proteomics 2004, 4: 3855-3863. There we used 10 uM prostaglandin I2, another PPAR gamma agonist. You can also check the paper by Rosenow et al. Journal of Proteomics 2010, 9: 5389 in which we used 7.6 uM rosiglitazone with human SGBS preadipocytes.
@Jin Cao: thank you for your advice. I'm starting the cell culture again using earlier passage, and I'm still waiting until they're ready for differentiation. I hope the problem can be solved this time.
Hi, I have done adipogenic differentiation of these cells using the protocol (in the attached paper), and got high percentage of differentiated cells (>80%). The protocol in the paper was not for 3T3-L1, therefore, the induction time was quite long (21 days in the paper). But for 3T3-L1, you can see lipid droplets in about 5 days, and the size increases day by day. Another thing brought my attention is, although many protocols mentioned that induction should start when cells are confluent, I found starting treatment when they are sub-confluent gave better result. Hope this helps.
I am new to this field. I read a lot of protocols are switching the differentiation medium to the medium with only insulin. Could anyone answer what's the purpose to do so? Thanks.
- Badges
- Science topic
- Similar topics
- Microbiology
- Assays