Authors
Creative Bioarray
Summary
The biochemical procedure is based on the activity of mitochondrial enzymes which are inactivated shortly after cell death. This method was found to be very efficient in assessing the viability of cells. A colorimetric method based on tetrazolium salts (e.g., MTT, XTT, CCK-8/WST-8) are especially useful for assaying the quantification of viable cells, because they are cleaved to form a formazan dye only by metabolic active cells. The formazan dye formed can be directly quantified using a scanning multi-well spectrophotometer, which enables on-line computer processing of the data (data collection, calculation and report generation) and, thereby, allows the rapid and convenient handling of a high number of samples.
Introduction
The succinate dehydrogenase in the mitochondria of living cell can reduce exogenous MTT to water-insoluble purple crystalline formazan and deposit in the cells, whereas dead cells do not. Dimethyl sulfoxide (DMSO) is capable of dissolving the formazan in the cells, and its absorbance can be measured at 490 nm by an enzyme-linked immunosorbent assay, which indirectly reflects the number of viable cells. The method has been widely used for the detection of biological active factors, large-scale anti-tumor drug screening, cytotoxicity test, and tumor radiosensitivity measurement.
Materials
Tetrazolium salt XTT has been developed for determining the number of viable cells in the medium. It is based on the reduction of XTT into a water-soluble orange yellow formazan compound by mitochondrial dehydrogenase. Since these enzymes are inactivated shortly after cell death, it is a reliable method for detecting viable cells. The absorbance of the formazan products can be measured directly from a 96-well plate without additional processing in combination with an electronic coupling agent such as PMS.
Procedure
CCK-8/WST-8 is reduced by the dehydrogenase in the cell mitochondria to the highly water-soluble yellow formazan product under the action of the electron carrier 1-methoxy-5-methylphenazine dimethyl sulfate (1-Methoxy PMS). The absorbance of the light is measured at 450 nm, which indirectly reflects the number of viable cells. The method has also been used for the detection of biological active factors, large-scale anti-tumor drug screening, and cytotoxicity test.
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