In cell functional experiments, the detection of cell proliferation, toxicity, and vitality is crucial. Currently, commonly used reagents for cell proliferation and activity detection are usually based on the reduction capacity of intracellular mitochondrial dehydrogenases, such as the MTT method, XTT method, WST-1 method, CCK method, etc.

Arcegen Biology has introduced a cell viability detection reagent kit based on WST-8 - Cell Counting Kit, abbreviated as CCK kit, which has lower cell toxicity compared to MTT, is more sensitive in detection, and easier to operate.

 

Experimental Principle

WST-8 (2-(2-Methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt) can be reduced by dehydrogenases in mitochondria to form highly water-soluble orange-yellow formazan in the presence of an electron-coupling reagent, 1-Methoxy PMS. The color intensity is directly proportional to cell proliferation and inversely proportional to cell toxicity. The OD value is measured at a wavelength of 450nm using a microplate reader, indirectly reflecting the number of viable cells.

Table 1. Comparison of Advantages of CCK Method with Other Cell Proliferation/Toxicity Detection Methods

Detection Method	MTT Method	XTT Method	WST-1 Method	CCK Method
Formazan Solubility	Poor (Requires dissolution in organic solvent for detection)	Good	Good	Good
Product Characteristics	Powder	2 bottles of solution	Solution	1 bottle of solution
Usage Method	Dissolve in solution before use	Ready-to-use upon preparation	Ready-to-use	Ready-to-use
Detection Sensitivity	High	Very high	Very high	High
Detection Time	Relatively long	Relatively short	Relatively short	Short
Detection Wavelength	560-600nm	420-480nm	420-480nm	430-490nm
Cell Toxicity	High, complete disappearance of cell morphology	Very low, no change in cell morphology	Very low, no change in cell morphology	Very low, no change in cell morphology
Reagent Stability	Fair	Poor	Fair	Very good
Batch Sample Testing	Possible	Extremely suitable	Extremely suitable	Extremely suitable
Convenience Level	Fair	Convenient	Convenient	Extremely convenient

 

Product Features

Ease of use: The product is a liquid and ready-to-use upon opening; formazan is water-soluble and does not require dissolution in organic solvents.

High sensitivity: Wide linear range, high sensitivity, and good repeatability.

Low cell toxicity: Very low cell toxicity, no impact on subsequent experiments.

Good stability: Presence of phenol red and serum in the culture medium does not affect detection.

Wide applicability: Suitable for large-scale, high-throughput sample testing.

 

Applications

Cell proliferation assays, cytotoxicity assays, drug screening, tumor drug sensitivity tests, activity detection of biological factors, etc.

 

Product Data

Comparison of performance with similar products

 

Cell type: 293T human embryonic kidney cells;

Culture medium: 10% FBS high-glucose DMEM medium;

Incubation time: 3 hours;Cell number: 1000

Referenced published literature data

GIST-T1 Cell Proliferation Assay CRTL represents the blank control group, Lv-shNC indicates the negative interference group, and Lv-shOrai1 denotes the Orai1 knockout group.	ROS17/2.8 Cell Proliferation Assay WT refers to the wild-type group, while KO represents the BK gene knockout group.
rMSCs Cell Viability Assay Cell viability assay under treatment with G/SWCNT hybrids, G, and SWCNTs at concentrations of 2.5, 5, 10, and 20 µg/mL.	rMSCs Cell Proliferation Assay Cell viability assay under treatment with G/SWCNT hybrids at concentrations of 2.5, 5, 10, and 20 µg/mL for 1, 3, and 7 days.
Detection of Huh6 Cell Interference on EZH2 Gene and Negative Control Group Cell Activity	Detection of HepG2 Cell Interference on EZH2 Gene and Negative Control Group Cell Activity
GIST-T1 Cell Viability Assay Cell viability assay under treatment with different concentrations of 2-APB (0, 50, 100, and 150µM)	GIST-T1 Cell Viability Assay Cell viability assay under treatment with different concentrations of SKF-96365 (0, 0.5, 1.0, and 1.5µM)

 

FAQ

Q1: How is the storage and stability of CCK?

A1: CCK has high stability. It is effective for 2 years at -20°C under light-avoiding conditions and for 1 year at 4°C. For frequent use, it is recommended to store at 4°C to avoid repeated freeze-thaw cycles. CCK is normally pink in color, and significant deviations in color may indicate changes in quality.

Q2: What is the cell toxicity of CCK?

A2: CCK has very low toxicity, allowing the same cells to be used for other cell proliferation tests such as crystal violet staining, neutral red staining, or DNA fluorescent staining after CCK detection.

Q3: Does CCK stain live cells?

A3: No, WST-8 does not enter the cells for staining. The entire color development reaction takes place in the culture system. Additionally, both WST and WST-8 formazan are highly water-soluble components, and replacing the culture medium with fresh medium after the reaction can remove exogenous components.

Q4: How many cells should be seeded per well for cell viability detection?

A4: When using a standard 96-well plate, the minimum seeding volume for adherent cells is at least 1,000 cells/well (100 μL medium). Sensitivity for detecting leukocytes is relatively low, so it is recommended to seed at least 2,500 cells/well (100 μL medium).

Q5: What is the usage of CCK reagent for experiments using 6-well or 24-well plates?

A5: For experiments using 6-well or 24-well plates, calculate the corresponding seeding volume per well and add CCK solution at 10% of the total volume of medium per well.

Q6: Will the background color of the medium, such as phenol red, affect cell viability detection?

A6: No, the absorbance of phenol red in the medium can be subtracted when calculating, eliminating its impact on detection.

Q7: Can 384-well plates be used for cell proliferation and viability detection experiments?

A7: Yes, the usage of CCK8 product is at 10% of the total volume of medium per well. It is recommended to dilute the CCK8 product twice and then add 20% of the total volume of medium per well to reduce errors.

Q8: Can OD values be measured only at 450 nm wavelength?

A8: OD values can be measured at 450 nm wavelength, but if there is no filter at this wavelength, a filter with absorbance between 430-490 nm can be selected. However, a 450 nm filter provides higher sensitivity.

Q9: What should be done if OD values are not being measured temporarily?

A9: If OD values are not being measured temporarily, 10 μL of 0.1 M HCl solution or 1% w/v SDS solution can be added to each well, and the culture plate should be covered and stored in the dark at room temperature. Measurement within 24 hours will not cause changes in absorbance.

Q10: Can the culture medium be not replaced when adding CCK-8?

A10: In general, the culture medium does not need to be replaced when adding CCK-8. However, if the culture medium contains redox substances, errors may occur, and other culture media must be used.

Q11: How to address high OD values during the experiment?

A11: You can reduce the incubation time after adding CCK-8. For example, you can shorten the incubation time after adding CCK-8 reagent from 2 hours to 1 hour. Additionally, you can reduce the number of cells appropriately.

Q12: How to address low OD values during the experiment?

A12: Two methods can be used: 1. Increase the number of cells appropriately. 2. Extend the reaction time after adding CCK-8 reagent.

Q13: Should a standard curve be done every time?

A13: It is recommended to do so. Although the cells are the same, their states may differ. For cells with different states, it is recommended to create a standard curve each time.

 

Related Products 

Product Name	Catalog Number	Specification
ClearV Cell Counting Kit (CCK-8)	C331301S	500 T
	C331301M	1000 T
	C331301L	3×1000 T
	C331301T	10×1000 T