Here we describe the real-time analysis of human stem cell differentiation using cell-based assays. This allows for quantification of cell viability, proliferation and apoptosis. 
Colorimetric Cell Viability Kit I (WST-8)
Colorimetric cell viability and cytotoxicity detection
The Colorimetric Cell Viability Kits I allows for easy and reliable colorimetric determination of viable cell numbers with excellent sensitivity and linearity. The kit utilizes the tetrazolium salt WST-8 that is reduced to water-soluble, orange formazan dyes by dehydrogenases present in viable cells. The absorbance of the formazan dye is proportional to the number of metabolically active cells and can be measured directly from a 96-well plate without additional processing.
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Technical Library (22)
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What is the purpose of the background wavelength? Do I have to use it?
It is recommended to measure the background wavelength; however, the background should be very low compared to the signal. Measuring the background confirms that there is no unexpected signal (for example, from the plate itself) that might interfere with the reading.
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How many cells should there be in a well when doing the WST-8 Viability Assay?
For adherent cells, at least 1,000 cells are necessary per well (100 µl medium) when using a standard 96-well plate. For leukocytes and other suspension cells, at least 2,500 cells/well (100 µl medium) are necessary because of lower sensitivity. The recommended maximum number of cells per well for the 96-well plate is 25,000. For 384 well plates, just divide these cell numbers by 4 (250 cells per well for adherent, 625 cells per well for suspension cells). If 24-well or 6-well plates are used for the assay, please calculate the number of cells per well accordingly, and adjust the volume of the WST-8 solution in each well to 10% of the total volume. For more exotic culture systems, like 3-D models or sphereoids, you should start with 10% WST-8 reagent per vol. of media and culture until you get a good reading. Some optimization of the protocol may then be necessary because the cells‘ metabolisms behave differently in 3-D.
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What is the difference between MTT, XTT and resazurin?
MTT (PK-CA-707-30006) and XTT (PK-CA20-300) are colorimetric based assays, while resazurin (PK-CA707-30025) 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.Related Links and Documents
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How many cells are required to obtain an efficient reading with the XTT Cell Viability Assay?
For most tumor cells, hybridomas, and fibroblast cell lines, we recommend 5,000 cells per well to perform proliferation assays, although as few as 1,000 cells per well have been used successfully. The known exceptions are blood lymphocytes, which require approximately tenfold more cells (25,000-250,000 cells/well) to obtain a sufficient absorbance reading.
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What is the principle of the MTT and XTT Viability Assay? How do they differ?
MTT and XTT Assays are both based on the conversion of tetrazolium salts to colored formazan derivatives. Tetrazolium salts are heterocyclic organic compounds that substitute the natural final electron acceptor (oxygen) in the biological redox process. Tetrazolium salts can penetrate rapidly into intact cells and directly into subcellular membranes. The assay principle is that – in living cells – MTT or XTT are reduced to formazan derivatives by either receiving electrons from substances of the hydrogen transport system or from artificial electron transporters. The amount of colored formazan directly correlates with the number of metabolically active cells.
While MTT is taken up by the cells through the plasma membrane potential and directly reduced inside of the cell, XTT (and WST) salts do not pass the cell membrane and are reduced externally at the cell surface via electron coupling reagents. The disadvantage of MTT is that the process includes the solubilization of formazan derivatives and that it is more cytotoxic than XTT (or WST) salts.
As an alternative to MTT, a new tetrazolium salt, XTT (2,3-bis(2-methoxy-4-nitro-5-[(sulphenylamino)carbonyl]-2H-tetrazolium-hydroxide), has been developed. XTT is converted into a water-soluble formazan, thereby avoiding the additional steps for the solubilization of formazan derivatives, but needs the presence of an electron-coupling agent (PMS) to enhance the reaction. The nature and the concentration of this agent are critical in order to obtain a good correlation between the formazan production and the number of viable cells.
Generally, the XTT (and WST) assays are significantly more sensitive than MTT assays and have a higher dynamic range. However, while the MTT assay shows good color development with almost all cell types, the XTT and WST assays might show weak signals with certain few cell types. Thus, in a very few cases, the MTT assay might be the better choice.Related Links and Documents
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The signal in my negative control sample (MTT, XTT, Resazurin Assay) seems to be high.
Serum and albumin in medium may lead to false positives in the MTT, XTT, and Resazurin Assay. It is best to use medium containing less than 10% added protein. Culture media containing up to 10% serum are usually fine.
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Is WST-8 (CCVK-I) toxic to cells?
CCVK-I solution itself is cytotoxic because of the high concentration of 1-Methoxy PMS. CCVK-I in the culture medium, however, is not cytotoxic because it is diluted by ten with culture medium. Therefore, long incubation, such as overnight and several days are possible. The same cell culture can be used for other cell viability/proliferation assays, such as the crystal violet assay or neutral red assay after the colorimetric WST-8 assay is completed.
Since each cell type has a different tolerance to CCVK-I, incubate cells with CCVK-I solution and check the cell viability if a longer incubation time is necessary.Related Links and Documents
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Are there cell types which will not work with the XTT Cell Viability Assay?
Cells with functional mitochondria are needed to convert the tetrazolium dye in its reduced form. Most eukaryotic cells in culture, including mammalian (suspension and adhesion), plant, and yeast cell types, reduce the dye sufficiently to perform accurate assays. But sometimes keratinocytes and melanocytes will show low metabolic activity.
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How does the XTT Cell Viability Assay work?
The XTT Cell Viability Assay is a colorimetric assay system which measures the reduction of a tetrazolium component (XTT) into soluble formazan product by the mitochondria of viable cells. The samples are read using an multiwell plate reader at a wavelength of 450 nm. The amount of color produced is directly proportional to the number of viable cells.
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How do XTT Cell Viability Assays results compare to [3H]-thymidine incorporation assays?
Because the XTT Cell Viability Assay requires less cell manipulation than [3H]-thymidine incorporation assays (no cell harvesting or medium changes are necessary), the possibility of errors is reduced and the standard deviation values are lower. Comparisons between [3H]-thymidine incorporation and XTT assays have demonstrated less than 5% difference for the determination of growth factor response.
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What is the purpose of the background wavelength? Do I have to use it?
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