Cell Counting Kit-8 (CCK-8) allows sensitive colorimetric assays for the determination of cell viability in cell proliferation and cytotoxicity assays.
CCK solution is a one-bottle solution; no pre-mixing of components is required. CCK, being nonradioactive, allows sensitive colorimetric assays for the determination of the number of viable cells in cell proliferation and cytotoxicity assays. WSTS* is reduced by dehydrogenases in cells to give an orange colored product (formazan), which is soluble in tissue culture medium. The amount of formazan dye generated by dehydrogenases in cells is directly proportional to the number of living cells.
Comparison between CCK method and MTT method:
1. Usually in the 96 hole plate, cell proliferation and cytotoxicity test are inoculated with cell suspension (5000cells and 2000 cells /100 uL/ hole, the specific number of the cells per hole should be decided according to the size and speed of the cell proliferation speed and other factors). The system is filled with a corresponding amount of cell culture medium, drug and CCK-8 solution, and the holes without cells are regarded as a blank control.
2. The cells adhered to the wall for 24 hours and are given a specific dose of 0-10 mg of stimulation in accordance with the experimental requirements.
3. The drugs are treated for 2~4 days and then add 10 uL of CCK solution at each hole (be careful not to generate bubbles in the pores, which will affect OD value)
4. Incubate the incubator for 1~4 hours, then use the microplate reader to determine the absorbance at 450nm.
5. If you do not detect OD value
temporarily, you may add 10 μL 0.1M HCL solution or 1% w/v SDS
solution, cover it with culture plate and keep it from light at room temperature. Absorbance does not change in 24 hours.
6. The absorbance of phenol red can be eliminated by blank hole so that the result will not be affected.
1. When using 96 hole plate to detect , if the cell culture time is longer,we must pay attention to evaporation problems. On the one hand, a circle surrounding 96 hole plate is more likely to evaporate, you can take it out, adding the same amount of PBS, water or liquid culture instead; On the other hand, the 96 hole plate can be placed near the incubator water to reduce evaporation.
2. The detection of this kit depends on the reaction catalyzed by dehydrogenase, so reducing agents (such as some antioxidants) will interfere with the detection.
3. If there are many reducing agents in the detection system, we should try to remove them.
4. Before using Microplate Reader, you should make sure there is no bubble in each hole, otherwise it will interfere with the determination.
5. This product is limited to scientific research of professionals and should not be used for clinical diagnosis and treatment or used as food and medicine, it should not be deposited in ordinary houses.
6. For your safety and health, please wear lab-gown and disposable gloves.
Cell viability * (%) =[A (dosing) -A (blank)]/[A (0 dosing) -A (blank) * 100%
A (dosing): Absorbance of cells, CCK solutions, and drug solutions
A (blank): Absorbance of the culture medium and CCK solution without cells
A (0 dosing): Absorbance of cells, CCK solutions without drug solutions
* Cell viability: Cell proliferation activity or cytotoxicity activity
4 degrees centigrades for one year; -20 degrees centigrades, protected from light for two years.
|Source||PLoS One (2013). Figure 3. Cell counting kit-8|
|Method||Cell proliferation assay|
|Cell Lines||Human colon cancer cell lines HT-29, LOVO, and Caco-2|
|Incubation Time||2 h|
|Results||The effect of cPA on cell proliferation was determined using a colorimetric assay. We found that cPA inhibited the proliferation of HT-29 and LOVO cells but not of DLD-1 and Caco-2 cells, which have lower endogenous levels of PDE3B than HT-29 and LOVO cells.|
|Source||PLoS One (2013). Figure 4. Cell counting kit-8|
|Cell Lines||Human mesenchymal stem cells|
|Incubation Time||3 h|
|Results||In group A, the cell number remained stable during the first 3 days, followed by a continuous increase till day 14. In group B, the cell number was stable during the first 2 days, then started to increase, and attained a plateau on day 12. In group C (control group), the cell number (Fig. 4A) remained stable during the first 4 days in culture, then started to increase, and plateaued on day 8.|
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