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Advantages and Usage of the CCK‑8 Cell Proliferation and Cytotoxicity Assay Kit
The Cell Counting Kit‑8 (CCK‑8 kit) is a rapid, highly sensitive colorimetric assay widely used for assessing cell proliferation and cytotoxicity, based on WST‑8 (chemical name: 2-(2‑methoxy‑4‑nitrophenyl)-3-(4‑nitrophenyl)-5-(2,4‑disulfophenyl)-2H‑tetrazolium monosodium salt). The assay principle is as follows: in the presence of the electron‑coupling agent 1‑Methoxy PMS, WST‑8 is reduced by certain dehydrogenases within the mitochondria to produce an orange‑yellow, water‑soluble formazan product (see Figure 1). The intensity of the formazan color is directly proportional to cell proliferation and inversely proportional to cytotoxicity. Absorbance (OD) is measured at 450 nm using a microplate reader, providing an indirect indication of the number of viable cells. This product is suitable for assays of cell proliferation, cytotoxicity screening, drug discovery, and evaluation of cell growth inhibition, among other applications.
Advantages of the CCK-8 Assay
Detection method | MTT assay | XTT assay | WST-1 assay | CCK-8 assay |
Product Characteristics | powder | 2 bottles of solution | solution | 1 bottle of solution |
Instructions for Use | Use after preparing the solution. | Prepare and use immediately. | Ready to use right away | Ready to use right away |
Detection sensitivity | High | Very high | Very high | High |
Testing time | Longer | Shorter | Shorter | Shortest |
Detection wavelength | 560-600nm | 420-480nm | 420-480nm | 430-490nm |
Cytotoxicity | High, cell morphology* has disappeared | Very low; cell morphology remains unchanged. | Very low; cell morphology remains unchanged. | Very low; cell morphology remains unchanged. |
Reagent stability | General | Poor | General | Very good |
Batch sample testing | Can do. | Very suitable | Very suitable | Very suitable |
Convenience level | General | Convenience | Convenience | Very convenient |
Transportation and Storage
Blue ice transport. Store at −20°C; shelf life: 24 months. [Note]: Repeated freeze–thaw cycles may lead to an increase in the assay’s background OD value.
Instructions for Use
Taking a 96-well plate as an example, 100 μL of cell suspension is seeded into each well, and the cells are pre-incubated in a cell culture incubator for 24 hours.
Add 10 μL of the test substance at different concentrations to each well. If only cell viability is being assessed, steps 2–3 are not required; proceed directly to step 4.
Incubate the culture plate in the incubator for an appropriate period of time (depending on the drug being tested).
Add 10 μL of CCK‑8 solution to each well (taking care to avoid bubble formation and gently mix).
Incubate the culture plate in the incubator for an appropriate duration (typically 0.5 to 2 hours).
The absorbance at 450 nm was measured using a microplate reader.
Precautions
This product is intended solely for scientific research and must not be used for clinical diagnosis, treatment, or any other purposes.
It is recommended to optimize both the number of cells seeded and the incubation time following the addition of the CCK‑8 solution. When using a standard 96‑well plate, seed at least 1,000 adherent cells per well (in 100 μL of culture medium) and at least 2,500 non‑adherent cells per well (in 100 μL of culture medium). Prior to the experiment, it is advisable to set up a series of wells with different cell densities to determine optimal conditions. Incubation time and treatment protocols should be tailored to the specific experimental design. After adding the CCK‑8 solution—at a volume equal to 10% of the total culture volume per well—incubate in a 37°C CO₂ incubator, and measure absorbance at 450 nm at various time points (CCK‑8 is highly sensitive; for some cell types, readings can be taken as early as 0.5 hours).
The CCK‑8 assay relies on a dehydrogenase‑catalyzed reaction; therefore, the presence of substantial reducing agents (or antioxidants) in the sample can elevate the background OD value and interfere with the results. If reducing agents are present in your experimental system, first measure the background OD and strive to eliminate their interference. For example, you may replace the culture medium with fresh medium prior to adding the CCK‑8 solution to remove the influence of the test compound. When the effect of the test compound is minimal, you can skip medium replacement and simply subtract the blank absorbance measured after adding the test compound to the medium.
During drug inhibition assays, the presence of metal ions—such as Pb²⁺, Fe²⁺, and Cu²⁺—in the test compound can interfere with the colorimetric reaction of the CCK‑8 solution, thereby reducing the assay’s sensitivity.
If the cells are cultured for an extended period and the culture medium changes color, wash the cells, replace the medium, and then proceed with the CCK‑8 assay. Phenol red in the culture medium does not interfere with the assay results.
To ensure thorough mixing of the CCK‑8 assay medium and minimize CCK‑8 residue on the pipette tip, it is recommended to dilute the CCK‑8 reagent with culture medium prior to aliquoting, and to mix well before dispensing.
If you do not measure the OD values immediately, you may add 0.1 M HCl or a 1% w/v SDS solution to each well, store at room temperature in the dark, and perform the measurement within 24 hours; the absorbance will remain unaffected. (The volume of the 1% SDS solution added should be equal to that of the CCK‑8 solution.)
If the absorbance is very low, you can appropriately increase the cell number or extend the incubation time after adding the CCK‑8 solution.
For your safety and health, please wear a lab coat and disposable gloves while performing this procedure.
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