MTT assay - Help needed (Mar/22/2006 )
Dear All,
I am learning the basics of tissue culture assays. I needed some help on MTT assay. I have read and learnt much about it. But most protocols suggest not to use media which contain phenol red during the assay. But the ATCC recommended media has phenol red. How can I get around this problem and have a good MTT result can any one help. It would of gr8 help indeed. Thanks in advance....
Naveen.
Wash your cells with PBS before MTT assay.
We incubate our cells sometimes in PBS containing MTT or sometimes in medium containing MTT. This medium does contain phenol red and the assay still works well.
Hi!
I also use medium containing phenol red, and I use the MTT dissolved in PBS and the assay works just fine. No problems so far!
People in our lab (another group) does MTT all the time and they grow cells in phenolred-containing media and have been doing so for years without any problem.
Your assay might become a bit more sensitive maybe, it depends on how precise you want everything to be...
The problem with the phenol red is it can absorb at the same wavelength as the MTT. Washing with PBS removes this problem.
Dear All,
Thanks for all your suggestion ideas and comments. This is what I can understand from this. After plating the cells and incubating them for attachment, one has to remove the media and then wash the wells with PBS and then add the MTT solution. Am I right? Or is it just add the MTT solution(dissolved in PBS) directly to the wells which has media? As many said phenol red may not affect my result that much. If some one can confirm this, I can happily go ahead and do my assay.
thanks all
I am learning the basics of tissue culture assays. I needed some help on MTT assay. I have read and learnt much about it. But most protocols suggest not to use media which contain phenol red during the assay. But the ATCC recommended media has phenol red. How can I get around this problem and have a good MTT result can any one help. It would of gr8 help indeed. Thanks in advance....
Naveen.
Do WST8 instead, and use your media as a blank.
Hi!
Like I said before, I always used media with phenol red and I never had any problem. After plating the cells and incubate them for attachment I add MTT dissolved in PBS (or in the same media used for the cells - the results are equivalent). But in each assay I include a blank (a well with no cells, just media and MTT). After making the measurements, I subtract the value of the blanks to the ones of the samples. That way you don't have to be afraid if phenol red interferes with the measure.
Hope it helps!
pals