The cell counting kit-8 (CCK-8) assay is a widely used calorimetric assay to assess cell viability and cytotoxicity. It is often applied multiple times on the same sample in tissue engineering and material research due to its low toxicity. It measures cell metabolic activity by detecting NAD(P)H levels through tetrazolium salt reduction, which reflects dehydrogenase activity. Because the formazan dissolves directly into the culture medium, the assay allows end-point measurement of living cell numbers.
Due to its high sensitivity and convenience, it is suitable for applications like cell viability assays, antimicrobial testing, and enzyme activity screening.
Principle of CCK8 assay
The CCK-8 assay is a highly sensitive detection method compared to other tetrazolium-based assays because it uses a highly water-soluble tetrazolium salt.
In this assay, the yellow-colored 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, sodium salt (WST-8) dye is reduced by the dehydrogenase enzymes present in the cells to produce a water-soluble orange formazan dye. This reaction involves an electron mediator called 1-methoxy-5-methylphenazinium methyl sulfate (1-m PMS). The quantity of this formazan product is directly proportional to the number of viable cells.
The reaction yields a distinct color change that can be quantified by measuring absorbance at ~450 nm using a microplate reader.
Reagents and materials
To perform the CCK-8 assay, the following reagents and materials are needed:
- CCK-8 reagent: The CCK-8 reagent is a ready-to-use aqueous mixture containing 5 mM WST-8, 0.2 mM 1-methoxy PMS, and 150 mM NaCl. While WST-8 is stable and not light-sensitive, it should still be stored in the dark and protected from air to maintain activity. Although some preparation is needed, the WST-8 assay is easy to perform and significantly more cost-effective. CCK-8 kit can also be used for this purpose.
- 96-well plates: The WST-8 assay is typically performed in 96-well plates with optimized reaction conditions, including buffer pH, incubation time, and reagent concentrations, to assess enzyme activities such as glutamate dehydrogenase (GDH) and glucose-6-phosphate dehydrogenase (G6PD) at specific temperatures.
- Microplate reader: Cell viability can be estimated by measuring the optical density of formazan at 450 nm using a microplate reader, with background absorbance subtracted from control wells lacking substrate. Although the CCK-8 assay offers a convenient colorimetric readout, it is limited to single time-point measurements.
- Pipettes: Sterile pipette tips are used to get reproducible results.
- Carbon dioxide incubators: Cell growth outside the body requires carefully controlled environmental conditions. CO₂ incubators are designed to provide these optimal conditions to support and maintain cell growth. They regulate and stabilize key factors such as temperature, humidity, and CO₂ levels to ensure consistent growth environments.
CCK-8 assay protocol (Step-by-step)
Here is a step-by-step CCK-8 assay protocol starting from the seeding cells to the analysis of the data.
- Seeding cells: CCK-8 assay can be used for adherent and suspension cells. Cell suspensions are carefully prepared and added to 96-well plates at a volume of 100 µL per well to ensure uniform cell distribution. Some wells receive only cell suspension to serve as controls, while others include test compounds to evaluate their effects on the cells.
- First incubation: The seeded plate is placed in a humidified incubator set at 37°C with 5% CO₂. Cells are incubated for the required exposure time to allow interaction with the test compounds.
- Addition of CCK-8 reagent: After incubation, 10 µL of WST-8 reagent solution is added to each well. The reagent is designed to react with dehydrogenases in metabolically active cells, leading to formazan color development.
- Second Incubation: The plate is returned to the incubator and incubated at 37°C for 2 hours. During this time, the WST-8 is reduced by cellular enzymes, producing a water-soluble formazan dye.
- Measurement of absorbance: After the incubation, measure the absorbance at 450 nm using a microplate reader. This absorbance correlates with the number of viable cells and provides quantitative data on cell proliferation or cytotoxicity. The reference wavelength is taken as 630 nm to correct optical artifacts.
- Calculation: Relative cell viability was calculated by comparing the absorbance ratio (A450/A630) of the experimental group to that of the control group. The absorbance of blank wells can be subtracted from all sample readings to obtain background-corrected values. Thus, the percentage of cell viability was calculated using the formula:
Percentage of cell viability = (Atreatment − Ablank) / (Acontrol − Ablank) × 100%
Storage and handling
The WST-8 solution is more stable than other dehydrogenase kits, remaining effective for up to one year when stored at 4 °C, unlike others that require −20 °C storage and expire within 2–3 months. Besides its high sensitivity, WST-8 offers advantages such as low cost per reaction, ease of preparation, and long shelf life. These features make it well-suited for high-throughput measurement of dehydrogenase activity.
Applications
CCK-8 assay can be used to test pharmaceutical products and environmental samples and evaluate cell toxicity and proliferation.
A study reported bacterial viability detection using CCK-8 assay by detecting live bacterial cells based on a redox reaction between the CCK-8 reagent and dehydrogenase enzymes present in metabolically active bacteria. Bacterial dehydrogenases reduce the CCK-8 solution to produce a water-soluble orange-yellow formazan dye. The amount of formazan, measured by absorbance at 450 nm, is directly proportional to the concentration of live bacteria.
Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were selected as representative gram-positive and gram-negative bacteria for method validation. Optimal assay conditions were established for accurate detection. A good linear correlation was observed between bacterial concentration and absorbance.
The CCK-8-based method showed reliable results when tested on real samples, aligning well with traditional plate counting. It allows for the simultaneous detection of many samples in a single run. Due to its high sensitivity and efficiency, the method holds strong potential for high-throughput bacterial detection.
Limitations of the CCK-8 assay
- The CCK-8 assay may be unsuitable for evaluating the cytotoxicity of colored drugs. This is because the color of the drugs can interfere with absorbance measurements, especially when the drug’s effect is subtle.
- Intracellular metabolic alterations can be detected in the CCK-8 assay. For example, a study reported the down-regulation of metabolites in glycolysis and the pentose phosphate pathway. These changes can affect energy metabolism and redox balance, so caution is advised when using CCK-8-treated cells in further experiments.
- Water-soluble tetrazolium salts, like those used in the CCK-8 assay, can be affected by Mg-, Mn-, Zn, or Cu-containing biomaterials, causing inaccurate cytotoxicity readings, leading to false negative results.
Conclusion
The CCK-8 assay has emerged as an essential tool for cell viability analysis in modern life science research. It is a straightforward protocol, based on the WST-8 tetrazolium reduction, allows researchers to obtain quantitative viability data with minimal effort and high sensitivity. It is a versatile and scientifically robust method that enables precise cell viability analysis, advancing research in cell physiology, pharmacology, and beyond.
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