We generally use the complete media containing phenol red to seed the cells in multiwell plate.Then after 24 h of culture we treat the cells with the drug containing media containing the drug and followed the MTT assay protocol for cell viability check.Here I want to know whether there is any effect of phenol red on MTT solution ?
Phenol red CAN be a problem! Phenol red and formazan absorption spectra overlap and you'll have phenol red absorption added to your formazan absorption readings. This is in itself would not be a problem if phenol red color was stable and not a pH indicator. However, it is not stable: if you treat cells with a drug that affects the metabolism, proliferation or whatsoever, this will affect pH of the culture medium (after 48hrs some of you observed changes in phenol red color..., guess why). And since these changes will depend on the treatment, you are not only measuring MTT-activity but also pH changes if you use phenol red medium.
The solution is simple: either you use medium without phenol red for such assays, or you follow the protocol of Tada et al (or similar)- here the SDS-solution to dissolve the crystals contains 0.01 HCl, making it acidic and when adding this to your cell medium any pH-dependent differences in phenol red color will be removed.
For more discussions or links to protocols:
https://www.researchgate.net/post/Which_is_the_best_method_to_solubilize_formazan_crystals_after_MTT_cell_labeling_in_proliferation_assays
It is better to use complete media devoid of phenol red, because phenol red may affect the MTT assay while taking the reading in ELISA reader
in my experience as long as you have your control wells with cells in same media , then phenol red causes no problems . adding to the fact that after 5 days incubation most of the phenol red color has changed anyway unless the cells have died. but when you calculate your CC50 you have your control cells and so any interference with phenol red is accounted for. If you can afford it i would recommend using MTS as it is much easier.
I have not observed any problems connected with phenol red and MTT reduction, yet. To be sure do a control test including MTT, drug and medium with and without phenol red and compare results.
Thanks to all.I will try with both the conditions (with phenol red and without).If we will get no difference its ok but if we will find difference then can we give conclusion that phenol red is affecting MTT.If yes then can any body suggest me a protocol to understand what exactly happened when MTT and phenol red is in together..
The problem with the phenol red is, it can absorb at the same wavelength as the MTT. So Washing the cells with PBS removes this problem.
Hi, I have the same question. What I'm doing is: seed the cells in media containing phenol red and after 24h add the drug. After that (more 24h) I will add MTT solution for 2-4h. So after that, should I take off the media, wash the cells with PBS and then add DMSO? Is that right?
Dear Puty, the protocol is similar to us but we never wash with PBS.
Phenol red CAN be a problem! Phenol red and formazan absorption spectra overlap and you'll have phenol red absorption added to your formazan absorption readings. This is in itself would not be a problem if phenol red color was stable and not a pH indicator. However, it is not stable: if you treat cells with a drug that affects the metabolism, proliferation or whatsoever, this will affect pH of the culture medium (after 48hrs some of you observed changes in phenol red color..., guess why). And since these changes will depend on the treatment, you are not only measuring MTT-activity but also pH changes if you use phenol red medium.
The solution is simple: either you use medium without phenol red for such assays, or you follow the protocol of Tada et al (or similar)- here the SDS-solution to dissolve the crystals contains 0.01 HCl, making it acidic and when adding this to your cell medium any pH-dependent differences in phenol red color will be removed.
For more discussions or links to protocols:
https://www.researchgate.net/post/Which_is_the_best_method_to_solubilize_formazan_crystals_after_MTT_cell_labeling_in_proliferation_assays
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