Im going to perform MTT assay on 3T3-L1 preadipocytes. After seeding cells in 96 well plate, do i have to wait 48 hours post confluent, then replace with treatment then only MTT solution? Or just 24 hours after seeding i can replace media with treatment and continue with MTT solution?
Hello Dayana Cheong
No, you do not have to wait 48 hours post confluent. Just 24 hours after seeding, you may replace the media with treatment media with the desired incubation time and continue with the MTT assay. 48 hours post-confluence is a condition to make the cells ready to be in a state to differentiate in the differentiation media as the cells start to get balled up and look a little bit uglier.
Best.
Thank you very much for your answer Malcolm Nobre . And the vehicle for my treatment will be the same complete media I used for maintaining the preadipocytes right?
Hi Dayana Cheong
Only wait 24 hours after seeding and you may replace the media with treatment media with the desired incubation time and continue with the MTT assay.
Hello Dayana Cheong
Yes, the vehicle for the treatment will be the same complete media you use for maintaining the preadipocytes. But you will have to include a vehicle control in the MTT assay.
For instance, if you have made the stock solution of your drug in DMSO and done further dilutions of the stock in culture media (the same media used to maintain the preadipocytes), then you will have to add only DMSO in culture media to the cells in one of the wells as the vehicle control.
Please note that the final concentration of DMSO should be less than 0.1% (in culture media) in the vehicle control as higher concentration of DMSO is toxic to cells. This is necessary so that you may be able to distinguish the effect of the drug treatment form the vehicle effect.
Best.
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