Introduction to Cytotoxicity Assay (CCK-8 Method)

What is Cytotoxicity?

Cytotoxicity refers to the direct cell-killing event induced by cells or chemical substances, independent of apoptotic or necrotic cell death mechanisms. Common cytotoxicity detection methods include the CCK-8 assay, MTT assay, and LDH assay.

Comparison with the MTT Assay

(1) The CCK-8 assay is more convenient to use and does not require cell washing.

(2) The CCK-8 assay enables rapid detection.

(3) The CCK-8 assay has a wide linear detection range and higher sensitivity.

(4) The CCK-8 assay shows better repeatability.

(5) The CCK-8 assay exerts low cytotoxicity and can maintain the original state of cells.

(6) CCK-8 reagents are relatively expensive.

Comparison with the LDH Assay

(1) CCK-8 is added directly to cells, whereas LDH is detected in cell supernatants, allowing the remaining cells to be used for other experiments.

(2) The LDH assay supports high-throughput detection.

Principle of Cytotoxicity Assay (CCK-8 Method)

The principle of the CCK-8 assay is briefly described as follows:

The CCK-8 kit contains the water-soluble tetrazolium salt WST-8 (chemical name: 2-(2-Methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt).

In the presence of the electron carrier 1-Methoxy PMS, dehydrogenases in the mitochondria of living cells catalyze WST-8 to produce a highly water-soluble orange-yellow formazan dye.

The amount of formazan produced is linearly proportional to the number of living cells.

Applications

1. Drug screening assays

2. Tumor drug sensitivity testing

3. Activity detection of biological active factors

4. Cell proliferation measurement, etc.

Materials and Instruments for Cytotoxicity Assay (CCK-8 Method)

【Materials】

Cell suspension, compound solution, CCK-8 reagent, complete culture medium, PBS, 0.25% Trypsin-EDTA.

【Instruments & Consumables】

96-well plate, carbon dioxide incubator, microplate reader with detection wavelength of 450–490 nm.

Procedures of Cytotoxicity Assay (CCK-8 Method)

1. Determine the seeding density in a 96-well plate according to the specific cell type, with reference to the control cell density reaching 70%–90% at detection. Set 3–6 replicate wells per group. Do not use the edge wells of the plate; add sterile PBS equal in volume to the culture medium to these edge wells.

2. Add CCK-8 solution at 1/10 of the culture medium volume per well. For example, if the initial culture volume is 200 μL, add 20 μL of CCK-8 solution, and scale accordingly for other volumes. Meanwhile, set blank control wells containing the same volume of cell culture medium and CCK-8 solution but no cells. If the test drug interferes with detection, use blank control wells containing equal volumes of cell culture medium, drug, and CCK-8 solution but no cells (avoid air bubbles).

3. Incubate continuously in the cell incubator for 2–4 hours. It is recommended to measure absorbance with a microplate reader at 2, 3, and 4 hours, then select the time point with appropriate absorbance range for subsequent experiments.

4. Measure absorbance at 450 nm. If a 450 nm filter is unavailable, a filter of 420–480 nm can be used. A wavelength greater than 600 nm (e.g., 650 nm) can be used as the reference wavelength for dual-wavelength detection.

Notes

1. This assay uses a 96-well plate. The outer wells are prone to evaporation, leading to inaccurate volume and increased error. Therefore, the outer wells are abandoned and filled with an equal volume of PBS, water, or culture medium.

2. Detection using this kit relies on dehydrogenase-catalyzed reactions. Thus, reducing agents (e.g., certain antioxidants) may interfere with detection. If a large amount of reducing agents exists in the test system, they must be removed.

3. Ensure no air bubbles are present in any well before measurement with the microplate reader, as bubbles will interfere with the assay.

4. Phenol red and serum do not interfere with CCK-8 detection; their background interference can be eliminated by subtracting the absorbance of blank wells.

5. The reagent is slightly toxic. Wear a lab coat and disposable gloves during operation.

6. Store the reagent protected from light at 4°C or -20°C.

Frequently Asked Questions

1. For slow-growing and small-sized cells, the maximum seeding density can be 1×10⁴ cells/well, but the maximum treatment time after compound addition is 72 hours.

2. If the compound is poorly soluble in water, DMSO or DMF can be appropriately added to the culture medium to aid dissolution. However, their maximum concentration should not exceed 0.5%, as DMSO and DMF also exert certain cytotoxicity. If DMSO or DMF is used for solubilization, ensure consistent concentrations of DMSO/DMF across all compound treatment groups.

3. What to do if absorbance values are too low?

(1) Appropriately increase the number of seeded cells.

(2) Prolong the incubation time after adding CCK-8 solution.