Greetings all,
My group has been trying to reproduce 3t3-L1 cell (preadipocytes) differentiation. The typical protocol involves 3 steps: the first step and second step both last for 2 days, and the third step varies in its time since it is more of a maintaining stage for the cells. This protocol is mature and used by many scientists, though details could vary by individuals, the general scheme is described as above. This protocol from University of Michigan is one of those we referred to: https://macdougald.lab.medicine.umich.edu/lab-protocols/protocolsmethods/3t3-l1-differentiation-protocol
We followed the protocol strictly. While other people can have most of the cells differentiated after 4 days entering the third step, we are not able to reach a similar result, in other words we have a low differentiation rate (since it is weird so we worry if it could possibly affect our further research?)
We tried to troubleshoot. For example, our cell passage number isn't too high, we prepare the differentiation cocktail correctly, our cells have no mycoplasma infection, we tried different serum but all these seemed not effective. We also tried using the same medium and serum used by other groups from our institute who can efficiently differentiate,though there is a problem that their cells have mycoplasma, so we could not say for sure, but still we weren't able to reproduce their results either.
In this case, should we consider that the problem is with the cell we are using? We bought it from ATCC. We also have contacted the distributor and asked for relevant information, but we didn't see any obvious problems.
I think we are lost, please share any thoughts that maybe helpful, thank you!
Could you post a picture of your cultures at 4 days? Does the % of differentiated cells increase after 4 days?
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