I’ve seen that some people remove the medium and others don’t. Others only add the MTT solution over the medium (with cells and treatment) and after 4h they add DMSO or isopropanol and read.
Can anyone explain what’s the difference between these two methods? and why they do it?
Which method do you recommend?
Hi Alvaro, I don't think remove medium or not is a question for MTT assay, because you have a negative control using the same setting. The final values will be normalized to your negative control. I once performed MTT assay using suspension cells, and I did not remove the medium.
Hi, Alvaro
Removing the culture medium causes the effects of various reducing materials to be eliminated and does not interfere with MTT color.Therefore, more realistic results are obtained.
Good Luck
I'm agree with previous responses - it better to remove media to do not have any problem with reducing agents and compounds what can affect mitochondria functions as well as membrane permeability during incubation with MTT.
I have tried both ways.
1. Without removing media, I added MTT. My results were fine. Here my drug (compound: some glycoconjugate) not interfere with MTT.
Recently, the same protocol didn’t work.
2. So, I have to remove the media, before adding MTT. Here my drug (some polyphenolic derivative). This is to avoid compound interference with MTT. I found that the compound (drug) reacting with MTT and producing the intense purple color with dose depend (from 100 uM to 500uM), where there are no cells and only compound and MTT.
So, removing media is the best option to get accurate results. But, be careful while removing media (there is a high chance that the cells may sucked while pipetting, then you may get random absorbance values). Hope this might be helpful.
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