MTT assay - (Jun/17/2009 )
I have looked at a number of protocols for the MTT viability assay and there seems to be much difference. These are;
1) Some protocols do not suggest removing the culture media before MTT addition. Just add MTT directly to media and then solubilise with DMSO.
2) Some protocols remove the media, then add MTT and then DMSO.
3) some protocols dont remove the media, add MTT to the media, but then remove all this (Media and MTT) and add DMSO.
Does anybody have a suggestion as to the best practise? I thought the MTT was uptaken by the cells, then cleaved to formazan and the formazan is returned by the cells to the media. This is then solubilised by the DMSO. Number 3 suggests the formazan is in the cells and not in media/mtt solution.
Any ideas would be a great help.
my method most closely resembles #3: I add MTT directly to media (0.5mg/ml final), incubate 90-120 minutes, aspirate the media with MTT, and lyse with DMSO. In my experience though, certain media formulations with high levels of ascorbic acid tend to result in some formazan accumulation in the media as well as in the cells, so I do PBS wash prior to the DMSO addition to be sure that I'm only measuring the product reduced in the mitochondria. You also don't want to over-extend the incubation times, since accumulation of the MTT precipitate can be somewhat cytotoxic.
The MTT is reduced primarily in the mitoch. to formazan, forming a precipitate inside the cell membrane that is not subsequently secreted. Usually very little formazan should accumulate in the media, unless theres an additive that interacts with the MTT directly.
That all said, there are MTT derivatives - MTS and XTT(I think)- that are supposed to produce a water soluble product that does not precipitate inside the cell and so is less toxic than MTT. But I've never used these myself, so .......?
I would recommend protocol No. 3. When I was doing cytotoxicity assays with MTT, I never replaced the media before adding MTT, unless the tested compound was coloured and would possibly affect the results. It always worked well. In the end I removed all of the cell supernatant and lysed the cells with DMSO or some other lysis buffer.
The use of XTT instead of MTT is also possible. We obtained the best results when we replaced the culture media with colouless OptiMEM prior to adding XTT. When using XTT you can measure the absorbance immediately after the incubation period without the need of cell lysis.
anemone on Jun 20 2009, 03:06 PM said:
I support that. We use XTT and it is pretty straight forward. We have the XTT resuspended in the colourless media, and we change it for a 2-4h incubation. We are still learning to use it, as somehow the general values are low, but there is a clear difference between the different cytotoxic compounds and when we rescue them. I do not see the advantage of using MTT anymore.