Based on my literature survey, I have seen that 24 hours for cell adhesion is recommended. The articles or standard video tutorials do not mention of attainment of morphology as an essential pre-requisite of MTT assay.
I am working with SHSY5Y which takes ~65 hrs to divide and more than 24hrs to attain morphology when seeding 15k in 96 well plate. I have been treating them with my stressor after 24hrs, and have been consistently getting no variation across Normal control and Treatment group.
The lack of variation can owe to the does of stressor, but my question is can the morphology matter significantly? I am treating them without attainment of morphology.
Hello Sanjukta Ghosh
No, not required.
The MTT assay is based on the conversion of MTT into formazan crystals by living cells, which determines mitochondrial activity. The MTT reagent can pass through the cell membrane as well as the mitochondrial inner membrane of viable cells, and only viable cells have active metabolism and can convert MTT to purple formazan via NADPH-mediated conversion.
So, you may consider MTT assay as a metabolic assay which only needs an intact and functioning mitochondrion. Please note that MTT assay is not a proliferation assay.
Best.
Thanks Malcolm Nobre
What did you mean by not being a proliferation assay?
Hello Sanjukta
I need to correct the last sentence. It should read as MTT assay is a type of proliferation assay that measures metabolic activity. There are other types of proliferation assays that measures ATP or DNA synthesis is also used as a cell proliferation marker.
Thank you for bringing it to my notice.
It is essential since, as you mention, the cells are adherent and, therefore, must be attached to the substrate to turn on their metabolism. Adherent cells unattached could die by apoptosis.
Hello Sanjukta
Adding to my above answer, see to it that the cells adhere to the substratum before you perform the assay. I agree with the above answers.
Some cells adhere within 8 hours while most of the cells should adhere within 24 hours. Once the cells adhere to the substratum you may perform the assay.
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