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 (23)
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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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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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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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What’s the principle of Caspase Fluorometric Assay Kits and Caspase Colorimetric Assay Kits?
These assays can be used to quantify specific caspase activities in cell lysates.
Assay principle:The assays use synthetic peptide substrates labeled with AFC (fluorometric detection) or pNA (colorimetric detection). Activated caspases in the lysates cleave their substrate, thereby releasing AFC or pNA, respectively. The more apoptotic cells and activated caspases are present in the lysate, the higher the fluorescence or absorbance signal measured.Related Links and Documents
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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.
Related Links and Documents
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Is there a correlation between WST-8 (CCVK-I) and the thymidine incorporation assay?
Yes. However, please note that since Colorimetric Cell Viability Kit I (CCVK-I) uses an assay mechanism (reduction of a tetrazolium salt to a colored formazan product by cellular dehydrogenases) different from that of the [3H] thymidine assay (measurement of DNA synthesis in actively growing cells), the results may differ.
Related Links and Documents
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What are the different types of multiwell plates that can be used in fluorescence, luminescence and colorimetric detection? Which type should I use for which application?
1) For fluorescence detection (fluorometer): Black plates with clear bottoms; often clear plates will suffice 2) For luminescence detection (luminometer): White/opaque plates 3) For colorimetric detection (photometer): Clear plates
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What type of biological sample do I need for the Caspase Assays?
Caspase Assays can be used with a variety of samples:
- The PromoKine Red and Green Caspase Staining Kits are suitable for live cells.
- The Caspase Fluorometric & Colorimetric Kits are suitable for cell lysates.
- The Fluorometric Assay Kits are also suitable for tissue lysates.
Related Links and Documents
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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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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.
Related Links and Documents
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What is the principle of the MTT and XTT Viability Assay? How do they differ?
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Synergistic anticancer effect of green synthesized nickel nanoparticles and quercetin extracted from Ocimum sanctum leaf extract
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Progranulin shows cytoprotective effects on trophoblast cells in vitro but does not antagonize TNF-alpha-induced apoptosis
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