Cell Function | Abnormal CCK-8 Experimental Results? This Article Will Help You Troubleshoot

Enhanced Cell Counting Kit 8 (WST-8/CCK8) is a rapid, highly sensitive, and non-radioactive colorimetric assay kit based on WST-8, widely used in cell proliferation and cytotoxicity experiments. The basic principle is that the reagent contains WST-8 [chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt], which is reduced by dehydrogenases in cells into a highly water-soluble yellow formazan product under the action of the electron mediator 1-Methoxy PMS (1-Methoxy-5-methylphenazinium methyl sulfate). The amount of formazan produced is directly proportional to the number of living cells. By measuring the absorbance at 450 nm, the quantity of viable cells can be indirectly calculated.

We can understand from the experimental principle that the CCK-8 assay results ultimately reflect as OD values. However, apart from the experiment itself, color changes caused by other factors can also affect the OD readings. Additionally, the CCK-8 assay mainly evaluates the total dehydrogenase activity in the sample, so any factor that influences dehydrogenase activity may lead to fluctuations in OD values. Therefore, improper handling during the CCK-8 assay can easily result in abnormal outcomes. Next, we will explore the potential abnormalities that may occur in the CCK-8 assay and provide corresponding troubleshooting strategies.

Significant Variability Between Replicate Wells

Apart from the impact of operational errors, the following factors may also contribute to significant variability between replicate wells:

1. Uneven Cell Seeding

1) Insufficient mixing of the cell suspension: If the cell suspension is not adequately mixed before seeding, or if cells settle during the mixing process, this can result in uneven cell distribution during seeding, potentially leading to significant differences between wells.

2) Improper seeding technique: During seeding, if the sample is added too quickly or the volume added is inaccurate, this can cause uneven distribution of cells in the wells of the plate. 

3) Differences in cell growth conditions: If the cells are in poor condition before seeding, such as aging, apoptosis, or variability in cell growth, this may also result in uneven cell distribution after seeding.

Optimization Strategies

①Ensure uniform mixing of the cell suspension: Before seeding, use techniques such as repeated gentle pipetting or gentle shaking to ensure uniformity in the cell suspension and prevent cell settling.

②Optimize seeding technique: Use a pipette or appropriate dispensing tools, angling them against the well plate walls to minimize errors between replicate wells. Control the seeding speed and volume to ensure consistent cell numbers in each well.

③Evaluate cell growth conditions: Before seeding, use an inverted microscope or similar tools to inspect the cells, observing their morphology and growth state. Ensure that fresh, healthy cells are used in the experiment, avoiding the use of aged or apoptotic cells.

Aggressive Drug Addition

Aggressive drug addiction can cause mechanical damage to cells, affecting their growth and proliferation capacity. This may result in varying numbers of viable cells between wells, ultimately leading to differences in OD values among replicate wells.

Optimization Strategy

Gentle drug addition: During drug administration, avoid aggressive handling as much as possible. Use appropriate tools like pipettes to slowly and evenly add the drug to the wells. Additionally, pay attention to controlling the speed and volume of drug addition to avoid creating bubbles or causing the drug to splash.

Liquid Evaporation

In microplates such as 96-well plates, the edge wells are more susceptible to external environmental factors (e.g., temperature fluctuations, air currents), causing them to evaporate more quickly than the inner wells. This can lead to significant differences in the growth environment of cells in the edge wells compared to other wells, resulting in what is known as the "edge effect," which affects the uniformity and reliability of the experimental results.

Optimization Strategies

①Pre-add medium or buffer: Before seeding cells, add an appropriate amount of medium or buffer (such as PBS) to the outermost two rows of wells on the plate to reduce liquid evaporation in the inner wells. Additionally, due to potential temperature differences caused by the incubator’s internal structure and airflow design, it is recommended to place the plate in the central position within the incubator.

②Reduce incubation time: Depending on the specific experimental needs and the growth characteristics of the cells, try to minimize the incubation time in the wells to reduce the cumulative effects of liquid evaporation.

Inadequate Mixing Before Detection

After co-incubating the CCK-8 reagent with cells, an orange-yellow formazan product would be generated, with the intensity of the color being proportional to the number and activity of the cells. If the liquid in the wells is not thoroughly mixed before detection, it may result in uneven concentrations of the formazan product in different areas, leading to inaccuracies in OD measurements.

Optimization Strategy

Thoroughly mix the liquid in the wells: After adding the CCK-8 reagent and incubating for the required time, gently shake or tap the plate to ensure the liquid in the wells is well-mixed. Take care to avoid generating bubbles, as they can affect the accuracy of OD measurements. If the liquid volume in the wells is large or the cell density is high, consider using a pipette to gently aspirate and dispense the liquid within the wells to further promote mixing. However, be careful to perform this operation gently to avoid causing damage to the cells.

Small Differences Between Groups

When the differences between groups are small, it may be due to the following reasons:

1. Excessive Cell Seeding Density

When the cell seeding density is too high, cells may experience "contact inhibition" due to the recognition effects of glycoproteins on the cell membrane and interactions between cells. This can cause the differences between different treatment groups to become less apparent.

Optimization Strategies

①Optimize cell seeding density: Determine the optimal cell seeding density through preliminary experiments. Generally, it is important to ensure that cells are neither too sparse nor too dense during cultivation. This will help maintain good cell growth conditions during culture and reduce errors caused by excessively high or low density.

②Improve cell culture conditions: Ensure that cells have sufficient oxygen, nutrients, and space to grow during culture. This can be achieved by regularly replacing the culture medium with fresh medium, using well-ventilated culture plates, and adjusting culture conditions as needed.

Drug Effects

①Impact of the redox properties of drugs: Certain drugs with reducing properties (e.g., Vitamin C) may react with CCK-8, leading to a color change that increases absorbance and results in artificially high measurements. This can obscure the true differences between groups.

Additionally, metal ions in drugs (e.g., zinc chloride, iron oxide, copper sulfate) may inhibit the colorimetric reaction of CCK-8, reducing detection sensitivity and making differences between groups less apparent.

②Direct effects of drugs on cells: Drugs may directly impact cell growth, proliferation, or metabolism. However, if the concentration of the drug or the duration of exposure does not reach a threshold that causes significant differences, these effects may result in only small differences between groups.

③Color interference from drugs: The color of the drug itself may be similar to or interact with the orange-yellow formazan product generated by the CCK-8 reaction, interfering with accurate absorbance measurement. Additionally, pH changes may cause the phenol red in the solution to change color, affecting OD measurements. These interferences may reduce the apparent differences between groups.

Optimization Strategies

①Drug pre-treatment: Before adding CCK-8, replace the culture medium with fresh medium to remove any drug-related interference with the CCK-8 assay. This step is particularly crucial when dealing with drugs that have redox properties.

②Optimize drug concentration and exposure time: Based on the drug’s characteristics (e.g., IC50 value) and the experimental goals, select appropriate drug concentrations and exposure times for the experiment. Preliminary experiments can help determine the optimal range for drug concentration and exposure time.

③Set up appropriate controls: By comparing the absorbance differences between experimental wells, negative control wells, and blank control wells, you can more accurately assess the drug's impact on cells.

3. Overextended Incubation Time

The CCK-8 reagent reacts with dehydrogenases in viable cells to produce an orange-yellow formazan product. As the incubation time increases, the accumulation of formazan gradually intensifies, deepening the color and raising the OD values. However, once the color reaches a certain intensity, it may hit a saturation point. At this stage, even if there are differences in cell viability, the changes in OD values may become less noticeable, thereby reducing the differences between groups.

Optimization Strategy

Optimize incubation time: Determine the optimal incubation time through preliminary experiments. In these preliminary tests, set various incubation time points (e.g., 30 minutes, 1 hour, 1.5 hours) to observe and compare the color changes and OD values across groups at different times. Choose a time point where the color change is noticeable but has not reached saturation as the incubation time for the formal experiment.

Mismatch in Trends

In addition to the previously mentioned anomalies, sometimes the trends observed in CCK-8 assay results may not align with trends from other experiments. This discrepancy could be due to the non-normal factors mentioned earlier, but it’s also possible that the results themselves are accurate. To understand this situation, it’s important to clarify which parameters the kit actually measures. In such cases, you can refer to our previously published articles:

The Data Doesn't Match? Correlation Analysis of Cell Cycle & CCK-8 Results

Mismatched Data? Annexin V & CCK-8 Correlation Analysis

We hope today’s discussion helps you navigate CCK-8 experiments with confidence. Accurate experimental procedures are key to obtaining reliable data. If you have more questions or would like to share your experiences, feel free to leave a comment!