Resazurin Cell Viability Assay
MTT and XTT are colorimetric based assays, while resazurin can be measured using colorimetric or fluorescence detection. MTT is not a soluble product, so the cells must be lysed to solubilize the formazan salt before absorbance can be measured. XTT and resazurin do not require cell lysis, allowing kinetic monitoring of the same samples at different timepoints.
AlamarBlue® contains resazurin and additional compounds to prevent the over-reduction of resazurin to a non-fluorescent product. These additives also slow the rate of generation of the fluorescent product. Consequently, the alamarBlue® assay requires longer incubation times compared to resazurin.
Resazurin is reduced by cells to the fluorescent product resorufin. Resorufin can be reduced further to a non-fluorescent compound. Therefore the densest wells may have lowest fluorescence due to over-reduction of the substrate. Please see the product information sheet for more details. The kit protocol provides general guidelines and may need to be optimized empirically for your experimental system. You may need to vary cell density or assay incubation time to ensure that your samples fall in the linear range of the kit.
Medium with phenol red is compatible with the resazurin assay. Phenol red does not interfere with the resazurin reaction nor does it affect detection.