i am performing MTT assay using lymphocytes. as lymphocytes do not adhere to the bottom of the well, so I cannot discard the media. I intend to add my cells(suspended in RPMI-1640) in each well. then I will add 100ul of my dilution( the chemical I intend to give exposure of, on the lymphocytes) to the well. the 96-well plate will be incubated for 24 hours. After that 1ul of MTT reagent is added to each well. After an incubation period of 4 hours, I have to add 100ul of DMSO. The problem is that after incubating my cells with the exposure substance I have to technically discard the media (but I cannot do that since the lymphocytes do not adhere to the bottom of the well, discarding the media would discard the cells too. What should I do?
Hello Mehar Khalil
You need not remove the media after incubating the cells with exposure substance unless and until your test compound should interfere with MTT color development resulting in an enhanced purple color.
To check whether your test compound will interfere in MTT assay, you will have to mix your test compound with the MTT solution in the absence of cells and verify whether this combination turns purple. If the color turns purple, it means that your test compound does interfere in MTT assay. In such a case, you will have to discard the media containing the test compound and rinse the cells before you add MTT solution.
To solve this problem, you should use round bottom 96-well plates because it will be much easier to get a cell pellet when you centrifuge these plates. In flat bottom plates, there are chances of losing cells when you pipette out the media as the cell pellet will not be compact.
You could centrifuge the 96-well plate at 300g for 5min for PBMCs and remove the media. You may/may not wash the cells. Resuspend the cells in MTT solution and incubate. Dissolve the formazan crystals in DMSO.
Best.
Hi, Yes centrifuge the cells , rinse per protocol and continue the test
You can use phenol red-free media and cut your media in half to 50µL/well then add 150-200µL DMSO. The residual media will not interfere with your measurements, plus you will have a media+drug well only so you can subtract it if you want and the DMSO doesn't get too diluted that you still get efficient cell lysis and dissolving of the formazan crystals. This is assuming your drug doesn't interfere with measuring the OD, which you will be able to determine in the media+drug with no cells well.
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