Cell Proliferation Reagent WST-1 From Roche Applied Science

The Cell Proliferation Reagent WST-1 by Roche Applied Science provides a colorimetric assay for the quantification of adherent or suspension cell viability and proliferation. The reagent is a sterile, ready-to-use solution that contains WST-1 and an electron coupling reagent diluted in phosphate buffered saline. WST-1 is a tetrazolium salt that when in contact with metabolically active cells gets cleaved to formazan by mitochondrial dehydrogenases. The formazan dye is then measured using a scanning spectrophotometer at wavelengths 420-480 nm. The darker the formazan dye, the greater the number of metabolically active cells in that well. This reagent can be used for a variety of experiments including: testing cell proliferation due to cytokines, growth factors, and nutrients; analysis of cytotoxicity due to anti-cancer drugs, pharmaceutical chemicals, or toxins. It is the latter that occurs in my lab where we use this reagent to test the cytotoxic effect of bacterial toxins and possible compounds that might reverse those adverse effects. This reagent has been utilized in my lab for approximately 1.5 years.

The WST-1 reagent has several advantages compared to the two other tetrazolium salt based cell proliferation reagents on the market. The MTT reagent is the traditional “old faithful” cell proliferation assay used today, but has a draw back due to the fact it is cleaved to a water-insoluble formazan crystal that must be solubilized before reading on the spectrophotometer. The WST-1, as well as the XTT reagent, is water-soluble after cleavage so there is no need to perform the solubilization step. WST-1 is more stable than the MTT and XTT assays so it is stored in a ready-to-use solution, unlike the XTT reagent where you must combine two reagents right before use. Also, the WST-1 reagent can be stored at 2-8ºC for several weeks without significant degradation. The specification sheet also says the WST-1 reagent has a wider linear range and accelerated color development compared to XTT. I’m not too convinced on this last statement, however, because our lab always used XTT before verifying data with the WST-1 reagent. After performing several experiments side by side with the XTT and WST-1 reagent, I did not notice an increased color development compared to XTT. The XTT reagent in my hands showed faster color development than the WST-1 reagent with MLg (mouse lung fibroblasts) cells as well as human lung epithelial cell lines, A549 and H441. The two reagents showed similar results in cell viability, however, so the variability is low between these two similarly based assays.

The general protocol for performing cell proliferation or cytotoxicity assays is simple and quick. Culture your cell line of interest into a 96-well plate with 100 ul/well final volume in a humidified incubator with 5% CO₂ and 37ºC. My lab uses 24-well plates with 200 ul/well even though the protocol calls for 96-well plates. Either plate layout works well. A cell concentration between 0.1 and 5x10⁴ and an incubation time between 24 and 96 hours is appropriate, depending on what cell line you are using. I suggest plating out your cells at increasing concentrations and time points to determine the optimal number of cells and incubation time to use. After determining how to plate your cells, perform the experiment you would like to test (i.e. challenge with toxins, test anti-cancer drug cytotoxicity, or affect of growth factors). Remember to test a blank well with no cells in order to subtract background absorbance from your experimental samples and controls. After the time points of your experiment are complete, add 10 ul/well of WST-1 reagent per 100 ul culture volume (20 ul/well for 200 ul culture volume). Place the plate back in the incubator for 0.5 to 4 hr. For the 24-well plates I utilize, I incubate for 0.5 hrs. Measure the absorbance using a microplate reader at wavelength 420-480 nm. Analyze the absorbance values to determine cell proliferation or cytotoxicity compared to control wells.

My lab has utilized the XTT reagent for several years before verifying the data with the WST-1 reagent. The reagents are very similar in the total time the experiment takes to complete and the ease with which the assay is performed. Even though the WST-1 reagent is more stable and is ready-to-use, since they are very similar we continue to perform the majority of our experiments with the XTT reagent because we have used it longer. I would recommend any lab already performing these assays to try the WST-1 reagent in your system to verify data and to determine if it is better for you. I would definitely recommend this over the MTT assay due to it being water soluble once cleaved and thus, no solubilization step is required. If this is the first time performing these kinds of assays, try the WST-1 first because of its increased stability and wider linear range.