I wanted to know if anybody has ever done MTT Assay outside of a 96 well plate before, possibly inside a larger container by increasing the volume of reagents used?
Our lab used to use a 48 well plate with fibroblast cell. I think it ok.But rather consuming reagents.
You can even do the MTT assay on the 12 well plate. But you must remember to increase the volume of MTT, to cover the well completely. During the measurments you must also remember to choose the reading in the proper plate (number of wells). Good luck!
I have experience with 24 well plate.MTT works well. If you are using large container you need to take a small sample after MTT from the container. better seed paralelly in any well plate AND CARRY THE WORK SIMULTANEOUSLY .
Hello
For 96-well plate use the protocol below :
1. Dilute the cells to 100,000 cells/ml and add 100 ul/well to a 96-well plate, then incubate 24 hrs at 5% CO2, 37C
2. After 24 hrs, remove all the medium and add designed compounds, incubate for 4 hrs
3. Remove all the compounds and add MTT solution (5mg/ml), 20 ul/well, then 80 ul of Medium (DMEM with 10 % FBS, 1% NEA and !% penicillin)
4. Wait for another 4 hrs of incubation
5. After 4hrs, centrifuge the plate and remove the supernatant, then add 50 ul DMSO to dissolve the formazan
6. Measure the plate at 570 nm
you can use DMEM, SDS and PBS as blank, negative and positive controls.
https://blog.quartzy.com/2017/05/01/cell-viability-assays-mtt-protocol
Good Luck
Dr Poorani Gananathan following your answer, do you find that your reading does become saturated sometimes? Since 24 well-plate can accommodate more cells, do you come across that you need to dilute the OD reading?
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