CCK-8 assay is a colorimetric method used to assess cell viability, proliferation, and cytotoxicity in various biological experiments. Cell viability refers to the number of healthy cells in a sample. In contrast, cell proliferation helps understand gene, protein, and pathway mechanisms affecting survival or death after exposure to toxic agents. Cytotoxicity and proliferation assays are essential in drug screening to evaluate test molecules’ effects on cell growth or toxicity¹.

Since drug toxicity is a major concern in pharmacology, it leads to the failure of many drug candidates and increasing development costs. Drug toxicity can be initially assessed through cell viability, which measures the proportion of living and dead cells in a sample. Developing reliable and scalable viability assays is vital for efficiently screening drug toxicity. A decrease in viable cells can result from inhibited metabolism or proliferation (cytostatic effect) or actual cell death (cytotoxic effect). Distinguishing between these processes is essential. Thus, the use of multiple methods helps accurately determine cellular changes².

Different assay methods are used to assess cell functions based on enzyme activity, membrane permeability, adherence, ATP production, co-enzyme production, and nucleotide uptake^1,3.

Principles of the CCK-8 assay

CCK-8 is a chromogenic indicator used to assess cell viability. Viable cells reduce yellow CCK-8 to an orange formazan product, with the intensity correlating to the number of live cells. This assay is effective for many cell lines, including non-adherent cells, within a range of 200-25,000 cells per well⁴.

Quantifying cell metabolic activity is essential for assessing viability. The CCK-8 assay measures this by detecting high nicotinamide adenine dinucleotide phosphate hydrogen (NAD(P)H) levels using tetrazolium salt, which reflects dehydrogenase activity. Since NAD(P)H is closely linked to metabolism, its depletion may impact cellular phenotype and function³.

CCK-8 is more sensitive to cell viability measurements than other tetrazolium salts like methylthiazolyldiphenyl-tetrazolium bromide (MTT), dimethylthiazol-carboxymethoxyphenyl-sulfophenyl-tetrazolium (MTS), and methoxynitrosulfophenyl-tetrazolium carboxanilide (XTT). Unlike MTT, it produces a water-soluble formazan upon reduction, eliminating the need for a dissolution or solubilization step. This makes WST-8 a more efficient and convenient choice for viability assays⁴.

Comparison with other cell viability assays

Mechanism of CCK-8 assay

WST-8 tetrazolium salt and its reduction process: The CCK-8 assay offers higher detection sensitivity than other tetrazolium-based assays due to its highly water-soluble tetrazolium salt. In this assay, WST-8 tetrazolium salt is used to measure live cells through bio-reduction of anabolic cofactors like NAD and NADP by cellular dehydrogenases. Active dehydrogenase enzymes in metabolically active cells reduce the tetrazolium salt that enters the cytosol. A water-soluble orange formazan dye is produced in the culture medium upon reduction. The amount of formazan formed directly correlates with the number of viable cells. The WST-8 solution is added directly to cells without any pre-mixing^5,6,7.

Function of electron mediator (1-methoxy PMS):  Electron mediator like 1-methoxy-5-methylphenazium methylsulfate (1-mPMS) assists in reducing WST to a formazan product with the help of NAD(P)H, with absorbance between 430-550 nm. The formazan absorbance is directly proportional to NAD(P)H concentration. This makes the WST-based colorimetric assay suitable for various qualitative and quantitative applications⁵.

Formation of formazan dye and its absorbance measurement at 450 nm: In standard culture conditions, viable cells convert the substrate into a detectable product, generating a signal proportional to their number. Dead cells lose this ability, forming the basis of many common cell viability assays. The absorbance pattern shows the viable cells, which peak at 450nm⁸.

Required materials and reagents

The CCK-8 cell viability assay requires the CCK-8 kit, laboratory equipment, storage conditions, and essential considerations to have non-contaminated and validated results.

CCK-8 kit components (WST-8 reagent and electron mediator)

The CCK-8/WST-8 assay kit is a convenient, no-wash, mix-and-read method for assessing cell viability. It uses a ready-to-use solution added directly to cell cultures. After 1-4 h of incubation, the number of living cells is measured using a colorimetric readout at 460 nm. The WST-8 solution’s low cytotoxicity makes it ideal for assays requiring long incubation periods (24-48 h)⁶.

Unlike other dehydrogenase kits that require -20°C storage and have a short 2-3 month stability, the WST-8 reagent is highly stable. It can be stored at 4°C for up to 1 year, making it more convenient and long-lasting. It is easy to use and significantly more cost-effective than other commercial dehydrogenase assay kits⁵.

Essential laboratory equipment

• Microplate reader: WST assays involve incubating a reagent with viable cells, leading to the conversion of a substrate into a detectable colored or fluorescent product. The reaction is performed in a microplate format. A single endpoint measurement is taken at 450 nm, using a plate reader⁸. The absorbance measurement shows the metabolic activity of viable cells. The absorbance is monitored in the extended range of 430 to 550 nm. At 450 nm it shows the maximum absorbance⁵.
• A CO₂ incubator: A humid carbon dioxide (CO₂) incubator creates a controlled environment that mimics physiological conditions. It supports optimal cellular growth by maintaining appropriate humidity, temperature, and CO₂ levels⁹. Human and mammalian cell lines or cell cultures are typically incubated at 37°C in 5% CO₂ incubator⁴.
• 96-well microplates: The WST-8 assay is performed in a 96-well plate with a total reaction volume of 150 μL⁵. Flat-bottom wells are recommended as they enable maximum light transmission, making them suitable for bottom-reading applications like high-content imaging. They are also ideal for growing adherent cell cultures as monolayers. Clear and optically transparent plates are necessary for proper light transmission during absorbance measurements¹⁰.
• Multi-channel pipettes for accurate liquid handling: A multi-channel pipette is recommended to ensure uniform reagent distribution and minimize differences between parallel wells. Consistency across all wells is a key challenge in microplate-based assays. Using a multi-channel pipette helps maintain accuracy and reproducibility in CCK-8 assays^11,12.
• Sterile pipette tips: Sterile and disposable pipette tips are essential for maintaining aseptic conditions in cell culture work. A new or thoroughly washed tip should be used for each well to prevent compound adhesion. This practice prevents cross-contamination, ensuring reliable and reproducible results¹⁰.
• Cell culture media: The cell culture medium should be suitable for the specific cell line used in the assay. Basal media lack essential proteins, lipids, and growth factors, requiring supplementation with FBS. To prevent biological contamination, antibiotics and anti-mycotics are often added to the media¹³.

Considerations for media composition and phenol red interference

Phenol red is a pH indicator commonly found in cell culture media. It absorbs light near the assay wavelength, which could interfere with readings. However, this absorbance can be subtracted as a blank and does not impact the assay results¹⁴.

Various methods solubilize the formazan product, stabilize color, prevent evaporation, and reduce interference from phenol red and other media components. Acidifying the solubilizing solution also changes phenol red to yellow, reducing its interference with absorbance readings⁸.

However, WST-8 is compatible with phenol red and other culture media, causing no interference⁴.

Storage

CCK-8 should be stored at 0-5 °C and protected from light. It remains stable for up to 1 year under these conditions. It can be kept at -20 °C for long-term storage, but repeated freezing and thawing may raise background signals and interfere with the assay¹⁵.

Experimental protocol for CCK-8 assay

• Cell preparation: Adherent cells are the cells that attach to the culture medium, while suspension cells are those that grow freely in the culture medium, showing non-adherence properties. CCK-8 assay can be used for both. The WST-8 reagent solution is an aqueous mixture. It contains 5 mM WST-8, 0.2 mM 1-methoxy PMS, and 150 mM NaCl. These components are dissolved in water to prepare the solution. Seed cells into a 96-well plate (100 µl/well) with or without test compounds; 200-25,000 cells can be seeded per well, although larger wells may be needed when using a higher density of cells and the volume of culture media and WST-8 reagent needed will need to be scaled appropriately, even for non-adherent cells⁴.

• Incubation conditions: For cells prepared in a 96-well plate with 100 µL per well, add 10 µL of WST-8 solution (frequently referred to as CCK-8 reagent) to each well, avoiding the formation of bubbles. Incubate at 37°C for 1-4 h⁶.

• Absorbance measurement and reference wavelength considerations: Measure the absorbance at 450 nm to 460 nm to assess cell viability⁶. The reference wavelength can be taken as 630 nm. Background subtraction can be performed using a reaction mixture without the substrate¹⁶.

• Standard curve generation: Measure absorbance values using the CCK-8 assay at 24, 48, 72, and 96 h after incubation to assess cell viability and observe color changes. You can then generate a standard curve by plotting OD values on the Y-axis against known cell counts on the X-axis. This curve can be used to determine cell numbers in unknown samples.

• Stopping the reaction and delaying measurements: To stop the reaction in a 96-well plate, either of the two methods can be used:

  • Method 1: Adding 10 µl of 1% SDS prepared in PBS, avoiding bubbles as they interfere with absorbance readings.
  • Method 2: Uses 10 µl of 0.1 mol/l hydrochloric acid, ensuring the reading is taken within 24 h, avoiding alkaline solutions such as WST-8 and similar unstable salts in such conditions.

Data analysis and interpretation

After the standard curve is generated, the data obtained is analyzed and interpreted to observe the viability of the cells.

Calculation of cell viability percentage

The mean optical density (OD) from the wells is used to calculate cell viability. Cell viability is then calculated by comparing the absorbance of treated samples to that of control (untreated) samples, typically using the formula:

Percentage of cell viability = (A_treatment − A_blank) / (A_control − A_blank) × 100%

Absorbance (A) represents the OD values measured. Final values can then be obtained by averaging (typically mean) results from duplicate wells for plotting¹⁷.

Use in proliferation assays and growth monitoring

The CCK-8 assay is extensively utilized to monitor cell proliferation under various experimental conditions. By measuring absorbance at different time points, researchers can generate growth curves that reflect cell proliferation rates.

For example, in studies involving vascular smooth muscle cells (VSMCs), the CCK-8 assay has been used to assess proliferation rates following genetic modifications or drug treatments. In one study, overexpression of miR-145-5p was found to diminish VSMC proliferation, as evidenced by reduced absorbance readings in the CCK-8 assay¹⁸.

Dose-response curve generation and IC₅₀ determination

In pharmacological research, the CCK-8 assay is instrumental in generating dose-response curves and determining the half-maximal inhibitory concentration (IC₅₀) of compounds. Researchers can plot dose-response curves to visualize the compound’s effect by treating cells with varying drug concentrations and measuring viability. The IC₅₀ value, representing the concentration at which the drug inhibits 50% of cell viability, can be calculated from these curves.

For example, in evaluating the cytotoxicity of anticancer drugs, the CCK-8 assay has been used to determine IC₅₀ values, providing insights into drug potency^5,19,20.

Importance of control wells for accurate interpretation

Incorporating appropriate control wells is vital for the accurate interpretation of CCK-8 assay results.

• Negative control: Control wells containing untreated cells establish baseline absorbance values,
• Background control: Wells with only culture medium help account for background absorbance.
• Positive control: Wells with known cytotoxic agents can serve as positive controls to validate the assay’s responsiveness.

Applications of CCK-8 assay

The CCK-8 assay has multiple applications in biological research. These include:

Drug screening and efficacy testing

The CCK-8 assay is commonly used in drug discovery to test pharmaceutical/therapeutic compounds. It measures how drugs impact cell viability. This information is important for determining both therapeutic effects and possible side effects²⁰.

CCK-8 assay is used to assess the cytotoxic effects of anticancer agents on tumor cells. A study compared this method with real-time cell analysis (RTCA) to test drugs like doxorubicin and curcumin on HeLa cells²⁰. The findings emphasized the CCK-8 assay’s reliability in drug screening applications. The CCK-8 assay is also suitable for high-throughput screening of large compound libraries²¹. It was effectively used in SARS-CoV-2 research to screen natural compounds for antiviral activity, showing its potential for therapeutic discovery²².

Cytotoxicity studies for environmental pollutants and nanoparticles

CCK-8 assays can determine the cytotoxicity of environmental pollutants and nanoparticles. For example, a study analyzed the cytotoxicity and protein expression levels of human B cell lymphoblastoid cells after exposure to cigarette smoke condensates (CSCs) from two different types of cigarettes.

The CCK-8 assay was used to detect these differences. Viable cell percentages ranged from 91.04% to 83.84% with one type of cigarette and from 90.2% to 12.28% with the other type of cigarette. Significant decreases in cell viability were observed at specific doses in both groups compared to controls²³.

Cell growth studies under varying conditions

A study comparing the cytotoxicity of various anticancer agents highlights the use of CCK-8 assay in determining IC₅₀ values for drug efficacy¹⁹.

Moreover, the CCK-8 assay has been used to investigate the effects of natural compounds on cell viability. In a study exploring the protective effects of Lycium barbarum polysaccharides against sevoflurane-induced neural injury, the assay demonstrated that these polysaccharides promoted cell viability and proliferation in a dose-dependent manner^23,24.

Specialized applications in tissue engineering and biocompatibility assessment

In tissue engineering, the CCK-8 assay helps in evaluating the biocompatibility and cytotoxicity of biomaterials intended for implantation. For example, a study assessing the biocompatibility of a 3D-printed hydroxyapatite/polycaprolactone (HA/PCL) scaffold used the CCK-8 assay to demonstrate that the scaffold supported the proliferation of bone marrow mesenchymal stem cells (BMSCs), indicating its suitability for bone tissue engineering applications²⁵.

Furthermore, the assay has been utilized to evaluate the cytotoxicity of novel biomaterials, such as chitosan/graphene oxide hydrogels, demonstrating enhanced proliferation and angiogenic capacity of endothelial progenitor cells, which are vital for effective tissue regeneration²⁶.

Limitations related to metabolic interference and enzyme activity influence

Despite its advantages, such as its availability as a ready-to-use solution in a single bottle, the CCK-8 assay has certain limitations. Compounds or materials that possess strong reducing properties, such as some metal ions like zinc (Zn) or manganese (Mn), can non-specifically reduce WST-8, leading to false-positive results. For instance, studies have shown that zinc-based materials like alloys can directly reduce tetrazolium salts, interfering with the assay’s accuracy²⁷.

Troubleshooting and best practices

• A pilot test can be conducted to identify the optimal cell number and incubation time.
• The presence of air bubbles in assay wells can lead to inconsistent absorbance readings, compromising data reliability. Pipetting slowly can prevent air bubble formation.
• CCK-8 reagent should be protected from extended light exposure. Repeated freezing and thawing should be prevented to decrease background signals and interference.
• Non-uniform mixing of the cell suspensions may affect the results. Uniform cell seeding is also vital for obtaining reliable results²⁸.
• Interfering substances like metal ions must be avoided to get the best results²⁷.
• If there is high turbidity (cloudiness or haziness), use a reference wavelength of 600 nm or higher to account for high turbidity in the cell suspension. The absorbance can be subtracted from 450nm to remove background noise.
• Proper controls should be used to get appropriate results after data interpretation.

FAQs

Can the CCK-8 assay be used for both cell proliferation and cytotoxicity studies?

Yes, the CCK-8 assay can be used for both cell proliferation and cytotoxicity studies. It measures the metabolic activity of cells, which increases with cell growth and decreases with cell damage or death. By comparing absorbance values, researchers can assess either the increase in viable cells (proliferation) or the reduction due to toxic treatments (cytotoxicity)

Why is CCK-8 considered safer and more sensitive than other tetrazolium-based assays?

CCK-8 is considered safer because it uses WST-8, which is non-toxic and does not require radioactive or organic solvents. It is more sensitive because WST-8 produces a water-soluble formazan dye with strong absorbance, allowing the detection of even small changes in cell number. Additionally, it enables continuous monitoring without harming the cells, unlike MTT or XTT assays.

Is CCK-8 toxic to cells, and can cells be reused after the assay?

CCK-8 is minimally toxic to cells because WST-8 is water-soluble and does not harm living cells during short incubation periods. This allows researchers to perform further analysis or reuse the cells after the assay if needed. However, prolonged exposure may still affect cell health, so timing should be optimized.

References

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