Roche’s cell proliferation reagent WST-1 is a water-soluble tetrazolium salt that can be used for cell proliferation or cell viability assays. The rate of WST-1 cleavage by mitochondrial dehydrogenases correlates with the number of viable cells in the culture. WST-1 is added directly to the cells (1/10th of the culture volume) and absorbance at 450 nm can be measured using an ELISA plate reader following a short incubation at 370C. The extremely simple protocol is ideal for screening large numbers of samples in 96-well plates. WST-1 is a safe and very simple alternative to tritiated thymidine uptake and is a significant improvement over XTT or MTS reagents. The advantages of WST-1 include safety, water solubility of the end product, stability of the reagent, scalability and sensitivity. WST-1 is provided with a practical and concise product information sheet and is featured in several product articles by Roche available online. In addition, WST-1 has been mentioned in more than 700 papers and used with numerous types of cells.
I successfully used WST-1 with primary mouse T-lymphocytes, lymphocyte cell lines EL-4, DO11.10, mouse fibroblast cell line L929 and human epithelial cell lines A549, HeLa and HepG2. However, WST-1 appeared to be unreliable with mononuclear phagocytes in my hands. The chemistry behind the assay suggests that the rate of WST-1 cleavage may be affected by the availability of NADH, redox status of the cells and/or the presence of reactive oxygen species. Therefore, the results from WST-1 assays should be validated by using alternative techniques. When using WST-1, care should be taken to check for formation of precipitates in the reagent stock. Incubation at 370C for 10 minutes followed by gentle agitation was always sufficient to resolve the problem. It is important to have at least one well with medium alone (without cells) for blank control. WST-1 added to the well should give background less than 0.2 absorbance units. The background value should be subtracted from the experimental wells for optimal results.
Although WST-1 has a relatively wide linear range, the signal could become saturated after prolonged incubation. The problem could be avoided by measuring absorbance at several time points for the initial experiments. I found that optimization of the incubation time was necessary for every new application or cell type. In addition, it is important to remove any bubbles from the culture wells. A short exposure to warm air (from a hair dryer) is very effective for that purpose. Following these simple precautions allowed me to perform quick and reproducible measurements of cytotoxicity of TNF-alpha for L929 and epithelial cells and activation-induced cell death in T lymphocytes.
Timur Yarovinsky, PhD
Associate Research Scientist
The University of Iowa