Hello, I hope you are all doing well.
I'm recently doing MTT assays to find the IC50 of my inhibitory drug. I have seen protocols that formazan crystals begins to form at 2 hours to 6 hours depending on the cell line you are working with.
I was wonder, can I add MTT treatment to my cells, leave them overnight in the incubator and the next day aspirate media and dissolve crystals with DMSO?
Thank you in advance.
Hello Eduardo Alvarez
It won't be right to leave cells with MTT treatment overnight. The conversion of MTT to formazan crystals by cells in culture is time dependent. Longer incubation time will result in accumulation of color and increased sensitivity up to a point. However, the incubation time is limited because of the cytotoxic nature of the detection reagents which utilize energy such as reducing equivalents like NADH from cells to generate a signal. Culture conditions that alter the metabolism of cells will likely affect the rate of MTT reduction into formazan. So, the incubation time for MTT in MTT assay is from a minimum of 30 mins to a maximum of 4 hours depending on the cell type and culture conditions.
Best.
Overnight incubation is not recommended, if you want to minimise your time of incubation, you can use freshly dissolved MTT for your experiment. HCT116 cells normally require about 2-3 hrs incubation with 1 month old MTT while it will take only 45mins to 1hr with freshly prepared MTT media.
Hi Eduardo Alvarez
In addition to the comments above, another reason is the toxicity of MTT at high concentrations. This results in all the samples eventually reaching the same concentration of MTT. Another issue is the limitations of the spectrophotometers used to measure absorbance - at very high concentrations, the machine will be unable to resolve differences in absorption due to the threshold limit.
On another note, MTT has started to become a little out-dated and there are plenty of other more sensitive and reliable products out there - for example, WST-8 is supplied in Abcam's CCK-8 assay kit and has the advantage of not needing to be dissolved in DMSO as the formazan produced is soluble!
https://www.abcam.com/cell-counting-kit-8-wst-8--cck8-ab228554.html
Good luck with your research.
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