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Breast Cancer Regulation
The measurement of cell proliferation and cell viability has become a key technology in the life sciences. WST-1 assay is much like MTT assay and the MTS assay, they are colorimetric assays for measuring the activity of enzymes that reduce MTT or close dyes (XTT, MTS, WSTs) to formazan dyes, giving an orange yellow. A main application allows assessing the viability (cell counting) and the proliferation of cells (cell culture assays). It can also be used to determine cytotoxicity of potential medicinal agents and toxic materials, since those agents would stimulate or inhibit cell viability and growth. An increase in cell proliferation is accompanied by increased signal, while a decrease in cell proliferation (and signal) can indicate the toxic effects of compounds or suboptimal culture conditions. This technique requires neither washing nor harvesting of cells and the complete assay from the onset of the microculture to data analysis by ELISA reader is performed in the same 96-well microplate.
WST-1 has several advantages compared to other cell proliferation agents such as XTT, MTS and MTT. In contrast to MTT which is cleaved to water-insoluble formazan crystal and therefore has to be solubilized after leavage, WST-1 yields water-soluble cleavage products like XTT and MTS which can be measured without an additional solubilization step. In contrast to XTT, WST-1 is more stable. And WST-1 has a wider liner range and shows accelerated color development.
• The appropriate incubation time after the addition of the WST-1 working reagent depends on the individual experimental setup (e.g., cell type and cell concentration used). Therefore, it is recommended to measure the absorption repeatedly at different points in time in a preliminary experiment. This allows you to determine the optimal incubation period for the particular experimental setup used.
• If precipitates or turbidity are observed upon thawing, warm up the solution to 37°C for 2-10 min and agitate to dissolve the precipitates.
1. Preparation of WST-1 reagent: add 5ml Electron coupling reagent into WST-1 powder and mix thoroughly. Incubate the solution for 2-10 min at 37˚C to dissolve sediments completely before use.
2. Collect cells on logarithmic phase. Count the cells and adjust the concentration of the cell suspension. Add 100 μl of a cell suspension (1000-10000 cells/well) to each well in a 96 well microplate. (Add sterile PBS buffer to marginal wells).
3. Incubate in a CO2 incubator at 37˚C until monolayer cells cover well bottoms (cells number of each well depends on cell size and proliferation speed). Add 0-10 μL drugs with concentration gradient in wells after cells adhere, usually two hours or half a day, including 5 duplicate wells.
4. Add 10 μL WST-1 Reagent to each well, and incubate for 4 hours. If drug reacts with WST-1, centrifuge and then remove culture medium. Wash with PBS buffer 2-3 times; add culture medium containing WST-1.
5. Optimal incubation time will depend on the individual cell type and concentration. For the first experiment, read results after 0.5 hour, 1hour, 2 hours and 4 hours incubation respectively, then chose the optimal incubation time for next step.
6. Shake plate for 1 minute on a shaker to mix contents.
7. Measure the absorbance using a microplate reader at 450 nm or at 420-480nm. Using dual wavelength spectrophotometry, you may choose wavelength longer than 600 nm.
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