Cell Counting Kit-8

Intended Use

Cell Counting Kit-8 (CCK-8) allows sensitive colorimetric assays for the determination of cell viability in cell proliferation and cytotoxicity assays.

General Description

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:

Protocol:

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.

Announcements:

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.

Vitality calculation:

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

Storage conditions:

4 degrees centigrades for one year; -20 degrees centigrades, protected from light for two years.

Documentation Download

Cell Counting Kit-8 Manual

Customer Product Validation

	Source	Sci Rep (2018). Figure 4. CCK-8 (AbMole BioScience)
	Method	Cell viability assay
	Cell Lines	PLC5 cells
	Concentrations	10 nmol/L
	Incubation Time	48 h
	Results	In accordance with functional analysis, western blot confirmed that CCRK overexpression promoted cell proliferation by activating β-catenin/TCF signaling, which however significantly abrogated by bufalin
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	Source	Sci Rep (2018). Figure 1. CCK-8 (AbMole BioScience)
	Method	Cell viability assay
	Cell Lines	PLC5 cells
	Concentrations	10 nmol/L
	Incubation Time	48 h
	Results	After 48 hours incubation, cell viability was measured by CCK-8 assay. In contrast with less influence on the growth of LO2 cells, bufalin exhibited strong ability to suppress the number of PLC5 cells in a dose-dependent manner.
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	Source	Cancer Biol Ther (2018). Figure 7. CCK-8 (Abmole Bioscience, Shanghai, China)
	Method	CCK-8 assay
	Cell Lines	C33A, Hcc94, HeLa, and SiHa cell lines
	Concentrations
	Incubation Time	72 h
	Results	As the results of the CCK-8 assay shown, the 72-hour treatment with buformin exhibited significant suppressive effects on cellular growth in C33A, Hcc94, and SiHa cells, but only exerted moderate effects in HeLa cells.
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	Source	Future Oncology (2018) . Figure 3. CCK-8 (AbMole BioScience, Shanghai, China)
	Method	Cell counting kit-8 assay
	Cell Lines	HeLa, SiHa, C33A and CasKi cells
	Concentrations	10 μl
	Incubation Time	2 h
	Results	Compared with its counterparts, knockdown of PXN suppressed cellular proliferation and induced cellular apoptosis
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	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
	Concentrations	10 μL/well
	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.
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	Source	PLoS One (2013). Figure 4. Cell counting kit-8
	Method	Cell viability
	Cell Lines	Human mesenchymal stem cells
	Concentrations	20 µL/well
	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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