Cell Proliferation Reagent WST-1 From Roche Applied Science

Roche’s WST-1 cell proliferation reagent is a simple, colorimetric assay designed to measure the relative proliferation rates of cells in culture. The assay principle is based on the conversion of the tetrazolium salt WST-1 into a colored dye by mitochondrial dehydrogenase enzymes. The soluble salt is released into the media. Within a given time period, the reaction produces a color change which is directly proportional to the amount of mitochondrial dehydrogenase in a given culture. As a result, the assay actually measures the net metabolic activity of cells. Theoretically, this is reflective of cell number – the more cells, the more dehydrogenase available to reduce the reagent. By the same reasoning, Roche suggests that the kit may also be adopted for use in measuring cell viability or cytotoxicity.

To perform the assay, the ready-to-use WST-1 reagent is simply added directly into the media of cells cultured in 96 well plates. The cultures are then given 30 minutes – 4 hours to reduce the reagent into the dye form. I found 30 minutes to be a reasonable time frame for osteosarcoma cell proliferation. The plate is then immediately read at 450 nm with a reference reading at 630 nm. Roche claims the dye has a larger linear range and increased stability compared to other tetrazolium salt based assays (e.g. MTS and XTT). However, I have not compared the WST-1 assay with these assays.

The WST-1 assay is suitable for use with adherent or suspension cells, although my experiences have been with adherent cells; specifically, osteosarcoma cells. Phenol red indicators in media do not appear to interfere with the dye reaction, although I would not recommend comparing WST-1 readings between phenol-red free wells and those with the indicator. For all media types, I recommend using a media-only well as a reference for the assay. The reference reading at 630 nm should further help to avoid the problem of background readings as it is subtracted from your original reading.

The major disadvantages lie in the concept behind the product. Although I briefly searched the literature, I was unable to find any data about the enzyme expression in the particular osteosarcoma cells I was using in my experiments. Therefore, I am unsure how much this enzyme can be regarded as a “house-keeping” protein, much in the way β-actin or other proteins are used as loading controls for Western gels. My experimental experiences with the kit would suggest the protein is not, in fact, ubiquitously expressed (or active) across all time points in culture. Following confluence, I noticed drastic changes (both up and down) of WST-1 absorbance over time. However, if the values are plotted on a logarithmic scale (as is common for growth curves) and enough repeats are conducted, reliable results can be achieved.

In this regard, it is best to test the assay with your cells of choice and conduct a time course cell growth curve experiment to determine the limitations of the kit with your cells. You may find your cells also drastically drop their WST-1 absorbance at certain stages of growth, and it would be best to keep future assays within the linear scale of growth as measured by the assay. As such, in my experience, a good general rule to follow is use the kit to measure proliferation differences in pre-confluent cells. The assay is extremely reproducible under these conditions and differences are generally a result of errors in cell seeding.

In addition, I would use caution in attempting to assay the proliferation rates of an entire 96-well plate. As the reaction is not stopped, it is feasible that readings taken towards the end of the 96-well plate have had an opportunity to further reduce the reagent than wells read first. Of course, you can reduce this error by adding the reagent in the order of and in sync with the reading time of your particular plate reader.

For best results, follow Roche’s guidelines regarding storage and handling. It is especially important to aliquot the reagent to ensure reproducibility of results, although once thawed an aliquot can be stored at 4ºC for some time.