Hello all,
I am using MTT assay to test the toxicity effect of a substance on A549 cells (adherent cell).
I seed 5x10^4 cells/well of a 96 well-plate, incubate overnight for cell attachment. Then I treated cells with the substance for 24hrs.
After treatment I observed that cells in the treated wells detached from the culture plate, changed the morphology to be round shape, and the cell confluency in treated well was significantly lower than control wells (observed under a microscope).
However, when used MTT to measured the cell viability, the treated wells gave higher signals than the control wells. I tried to observe the well after incubating with MTT for 2hrs, I saw the cells in treated well had more MTT crystals in the cytoplasm than those in the control wells.
Is it false positive data? How could I solve this problem?
Thank you very much for your help.
It can happen that a substance can by itself reduce MTT to formazan crystals, so you should always use a control without cells, but with your substance.
MTT is assay that dont need to give true picture of viable cells! It is reduced in mitochondria and if the metabolism of cells is somehow increased those mitochondria are capable to convert larger amount of MTT than those in control. For more info on this you can read the original reference (Mosman).
Even when the cells are detached, if the mitochondria is functional, it will give positive results. You should check the possible activity of your substance on the junctions and network created by cells to adhere to the wells.
As Mr. Stojanovic said, MTT is just a preliminary method of testing cytotoxicity. You shouldnt completely rely on the assay. Try performing a FTIR analysis to determine what kind of surface groups in your substance are inducing this kind of result.
You treatment probably works on mitochondria inducing their activity and their capabilities to convert MTT to tetrazolium.
You could perform an assay of mitochondrial activities or move to a different viability test such as sulphorhodamine B (Sulforhodamine B colorimetric assay for cytotoxicity screening, Nature Protocols 1, 1112 - 1116 (2006) doi:10.1038/nprot.2006.179)
It can happen that a substance can by itself reduce MTT to formazan crystals, so you should always use a control without cells, but with your substance.
Thank you all for your helpful suggestion.
I also would like to try Calcein-AM assay. Has anyone performed that assays? Could you please give me some opinions? Is it more sensitive than MTT and LDH assay?
Thank you again!
Hi- that is really interesting as I have had the exact same experience with a WST-1 assay (a type of MTT). When I treat dermal fibroblast cells with a drug of interest I have visibly less cells down the microscope but equal or higher WST-1 result. I am currently undertaking some further investigations with Mitotracker to see if there is any onvious change in mitochondrial number or morphology and maybe possibly look at nuclear vs mitochondrial DNA copy number to see whether there could be a metabolic change in these treated cells...
Thank you sir Robert!
Hello Rachel,
So interesting! I also would like to know the results of your on-going experiments. Please tell the the results of your further investigations.
Thank you
Yes, this is really interesting - we've sometimes had a similar result to Rachel with primary fibroblasts with the MTT assay, in that drug treatment results in visibly fewer cells but they are more strongly coloured so it's difficult to derive an EC50. From what I remember, our experiments with primary keratinocytes gave similarly erratic results. I'd like to look at alternative assays to try and investigate this further, e.g. SRB or EdU assays, although Mitotracker could be informative too. It seems this kind of problem could be quite common.
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