Hello Mohammed I. A. Ibrahim

There are certain reductants present in cell culture media, such as free thiols, which can reduce MTT under basic conditions. Furthermore, the reduction of MTT by these compounds is influenced by the type and pH of the medium used. Common reducing agents like reducing sugars, ascorbic acid, and oxidation-prone sulfurous amino acids that are present in cell culture media, are generally used in assays, which may cause unforeseen interference.

For instance, cysteine and methionine can be easily oxidized which contain a thiol and a sulfide functional group respectively, as well as glutathione (GSH), a tripeptide containing a free thiol and ascorbic acid which is used as an antioxidant, as well as glucose, a reducing sugar.

Through the mechanism of thiol-mediated redox reactions of MTT, ascorbic acid, cysteine and glutathione (GSH) are reactive with MTT under basic conditions. Therefore, when performing MTT assay, reducing agents which are common to cell culture media may alter MTT results.

To combat this issue, you either use reducing agent-depleted culture media to conduct the assay to ensure that MTT assay is carried out with minimal interference or use a higher cell density for MTT assay to prevent interference through auto reduction by reducing agents in culture media, or you may use a blank well in MTT assay consisting of a cell-free culture medium plus an MTT solution and a solubilizing solution. The blank well represents the background absorbance value created by the medium itself that must be subtracted from final values of the samples, positive and negative controls.

I have attached an article below which will be helpful!

https://emerginginvestigators.org/articles/22-186/pdf

Best.