First Edition (Revised on April, 2016)
WST-8 [chemical name: 2-( 2-methoxyl-4-nitrophenyl )-3-( 4-nitrophenyl) -5-( 2,4-dimethyl-Benzenesulfonic acid )-2H
-Tetrazole monosodium salt], is restored to highly water-soluble yellow formazan product by means of 1-Methoxy PMS. The number of formazan product is proportional to the number of living cells. The CCK-8 kit is based on WST-8 coloration, which is widely used in rapid and high sensitive detection of cell proliferation and cytotoxicity.
[REAGENTS AND SPECIFICATION]
Drug screening, cell proliferation test, cytotoxicity test, tumor chemosensitivity test.
[STORAGE AND DEADLINE]
Store at 4 degrees, and the the shelf life is 12 months.
1. Prepare a certain concentration of cell suspension from well-grown cells, 100ul per well in 96T cell culture plate. According to the experimental requirements, add 0-10ul drugs into the hole and continue culture.
2. Add 10ul CCK-8 solution to the 96 hole cell culture plate and incubate at 37degrees for 0.5-4 hours.
3. Determine absorbance at 450nm. Recommended dual wavelength determination, the detection wavelength is 430-490nm and the reference wavelength is 600-650nm.
1. Generally, cell proliferation test would add 2000 cells/100ul in every well, and the cytotoxicity test would add 5000 cells/100ul in every well. (The number of cells used depends on the size of the cells, the speed of cell proliferation, and so on.)
2. For adherent cell, it is necessary to preculture it at 37 degrees for 2-4 hours. Until cells stick to wall, the experiment can be started. For suspension cell, it doesn't have to be precultured.
3. For cytotoxicity test, the incubation time after adding poisonous substance needs to be develpoed. It depends on character of poisonous substance and cell sensitivity and is generally determined by the cell cycle, at least for a generation.
4. If the initial cell culture volume is 200ul, then need to add 20ul CCK-8 solutions, and so on.
5. Setting up 3 replicating holes for each drug concentration, and then take the mean.
6. Design control to exclude other factors: 1）blank control: add equal cell culture fluid, CCK-8 solution and drug without cells. 2）negative control: the hole without drugs but with a solvent.
7. The kit depends on the dehydrogenase catalysis reaction. If the material contain oxidation or reducibility, it may disturb the detection and need to remove it. ( Replace fresh medium before adding CCK-8 to remove drug effect. If the effect is small, there is no need to change the medium, take blank absorption after the drug is added out.
8. Pay attention to the incubation time after adding CCK-8. Usually incubate for 1 hour and the incubation time depends on the type and density of cells, and so on. In the first experiment, can be detected by ELIASA at 0.5,1,2 and 4 hours, and then choose a proper time for the follow-up experiment.
9. When incubated in incubator, the outermost well is easy to dry and volatilize, which will cause error. In general, the outermost hole only adds medium, but it doesn't serve as a measuring hole.
10. If the OD is not determined at once, add 10ul 10%SDS to stop reaction. Cover culture plate, store at room temperature and avoid light. There is no obvious change in 24 hours, but it is generally recommended to check immediately.
11. Make sure that there are no bubbles in each well before testing by ELIASA, otherwise it will disturb determination result.