**2. Colorimetric assays with tetrazolium salts**

In colorimetric cytotoxicity methods, measurements are made based on color change upon addition of tetrazolium salts. Tetrazolium salts are heterocyclic organic compounds. The reduction of tetrazolium salts by gaining electrons enables them to transform into a formazan structure and leads to a color change. The tetrazolium ring can be broken by active mitochondria, and therefore, color change can only occur in metabolically active cells. Taking advantage of these properties, many tetrazolium compounds have been developed [4]. However, only a few of the tetrazolium salts have been accepted and adapted to biological systems. The most widely used tetrazolium salts are listed below:


These compounds can be divided into two groups based on their ability to enter the cell and dissolve in the medium. MTT sits in one of these groups that the tetrazolium salt that has the ability to enter the cell. This salt is a positively charged compound and can be reduced within the cell by conveniently passing the membrane of eukaryotic cells. However, the formazan from reduction is insoluble in water and therefore precipitates in the medium as crystals. Unlike MTT compounds, MTS, XTT, and WST compounds are naturally negatively charged and cannot fully penetrate the cell membrane. Therefore, they use an electron-accepting molecule with themselves. The electron acceptor molecule enters the cell, takes electrons from the cytoplasm or plasma membrane, and returns to the medium to reduce the tetrazolium compound. Formazan, which is formed as a result of the reduction of these compounds, is soluble in water and the medium. Phenazine methyl sulfate and phenazine ethyl sulfate are generally used as electron acceptors in these reactions [5].
