When I perform an MTT assay after adding DMSO, only a brown colour is appearing. I am working with suspension cells and I want to know the cytotoxicity of a compound. After 24 hours of seeding cells in 96 well plate, I am treating cells, and incubating for 24 hours. Later, I remove the medium and add MTT, and after 2 to 3 hours remove MTT and add DMSO. Why I am not getting purple colour? Any suggestions as per the problem, and any potential solutions?
Is your MTT freshly prepared? It oxidizes quite fast and should be prepared directly before adding. What protocol do you use? Are you preparing stock solution?
Instead of DMSO try adding isopropyl alcohol.
For 100ul of suspended ceels I'm adding 12ul of MTT (without removing medium) and after 1-2 hours I'm addind 110ul of isopropyl alcohol.
And it always works :)
Good luck
I agree with Anna. How was the assay kit stored? Have you followed the protocol in mixing the agents?
Was it your first ever cytotoxicity test?
Have you got enough cell numbers in each well?
What were the responses for positive and negative controls?
DMSO has peroxides in. Need to use cell culture grade. Even then it will slowly accumulate peroxides.
Perhaps your MTT is oxidized, just like what others said above. You should use fresh reagents in any type of assays, especially colorimetric tests like MTT cytotoxicity test.
Or, your cells might produce some substances that reacts with the MTT. But this is just purely my opinion.
Allan is right. You should use fresh reagent colorimetric test. With MTT you should prepare stock solution and storage at -20 before use for the test.
Before apply test solution to the cell, you should check cell density; it survive or not; the density of cell is enough or not. Good luck
I believe for Suspension cells you need to ensure that when you drain medium there are cells left or you are draining that too.
If there are no cells then MTT wun give purple color.
Some labs use Spectramex centrifuge to retain cells before passaging or draining our medium. Plz check possibilities in your set-up...
I have always used the Promega MTT kit, it always works and you don't have to worry about removing medium, also, theirs MTS kit is better than MTT, no need to kill the cells and you have a result in 4 hours.
Thats True Gisela., medium need not to be removed, by removing medium cells are removed too, as being suspension cells.
As others have said, you don't need to remove the media.
I'm not sure whether it could be due to the DMSO as we have always used a THF/SDS solublizer with no problems in either adherent or suspension grown cells.
Also, we have always stored our MTT solution at 4 degree in the dark, this is good for months in our hands.
As you know the only reason to solubilize the cells is to be able to quantify colour, you will see brown/purple cells before the addition of the solubilizer.
Does your media only blank stay yellow?
Do your cell only controls turn brown?
If, yes to both of these then it doesn't matter if they are brown. Brown and purple will both absorb at 570nm.
If you can distinguish between positive (known cytotoxin) and negative (carrier solvent only treated) on the plate reader the exact colour does not matter. I don't think you have a problem really
BTW, why are you seeding the plate 24h before you are adding the drug? Most of the time you only need to do this with adherent cell lines to allow them to attach.
Even if you are doing short time exposure to the drug, all you need to do is adjust the cells to the appropriate density (the density that gives you ~90% maximal MTT staining after the drug exposure time period but with no drug added), seed the plate and you can add drug straight away.
Instead of DMSO, add a solution containing 10 % SDS and 40 % dimethylformamide in H2O, pH = 3,9. Maybe the purple color appears pH-dependently. I also agree to not remove the medium. I use cells in 100 micorliters culture medium, add 10 micorliter MTT (5 mg/ml PBS) which was stored at 4 °C in a dark flask, let cells metabolize it for 3 h and then add 100 micorliters of the buffer! Good luck!
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