I'm using Sigma reagent for MTT test. In our protocol we add MTT, incubate for 3h and solubilise with acidified isopropanol, by adding it 1:1 to the wells, and mixing properly, without removing the medium.
I read there are other protocols where you remove all the medium after incubation with MTT, and use DMSO or SDS.
I am interested to know the reasons for bothering with removing the medium but more specifically the pros and cons of using medium.
some people are removing the medium becoz they want the cell lysis faster and the blue color come out faster and easily can read the absorbance. but other researchers believe removing the medium is disturbing the cells and some of the cells migh go out from the well and that cause error in your assay. Anyway I suggest you to your MTS, is more convinence and you dont need to use Isopropanol or DMSO to stop the reaction. once you add MTS after 1 hor to 4 hour you can detect your plate using ELISA reader.
If you are using semi-adherent cells or non-adherent cells than it's harder to remove the media following incubation with MTT (you would need to spin down the cells first). In this case it would be easier to use the MTS assay, as mentioned above.
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