Assaying Cell Proliferation and Viability with Cell Counting Kit 8 (CCK-8)

Cell Counting Kit 8 (CCK-8) is a sensitive colorimetric assay used in biomedical research to determine cell viability, proliferation, and cytotoxicity. It works by employing the highly water-soluble tetrazolium salt, WST-8, which is reduced by intracellular dehydrogenases in live cells into a water-soluble, orange-colored formazan dye. The intensity of this color, measured by a microplate reader at a specific wavelength (around 450 nm), is directly proportional to the number of metabolically active cells.

The CCK-8 assay offers ease of use, minimal toxicity, and enhanced sensitivity compared to tests based on other tetrazolium salts such as MTT, XTT, MTS, or WST-1.

Cell Number Determination

To enable the quantification of absolute cell numbers in subsequent assays, a standard curve correlating the optical density (OD) with the cell count must be established under consistent experimental conditions.

Count the cell number in the suspension using a cytometer. Seed cells into a 96-well plate at the calculated density.
Serially dilute the cell suspension in culture medium to generate a gradient of cell concentrations. A minimum of 3 to 5 distinct concentrations is recommended; each replicated in 3 to 6 wells.
Culture cells until they have adhered to the wall. Subsequently, add CCK-8 reagent to each well and incubate for the predetermined duration.
Measure the absorbance at 450 nm.
Construct a standard curve by plotting the mean OD value (Y-axis) against the corresponding known number of seeded cells (X-axis). This curve allows the determination of the cell number in an unknown sample cultured at identical assay conditions (e. g., incubation time post-CCK-8 addition, etc.).

Cell Viability Assay

Seed cell suspensions (100 µL/well) in a 96-well plate.
Pre-incubate the plate for 24 h in an incubator at 37 °C and 5% CO₂.
Add 10 µL of CCK-8 reagent to each well, avoiding the introduction of air bubbles, as they can interfere with absorbance readings.
Incubate the plate for an additional 2 h.
Measure the absorbance at 450 nm using a microplate reader.
(Optional) If the absorbance cannot be measured immediately, the reaction may be stabilized by adding 10 µL of 0.1 M HCl or 1% (w/v) SDS solution to each well. The plate should then be covered and stored in the dark at room temperature; the absorbance values remain stable for up to 24 h under these conditions.

Cell Proliferation and Cytotoxicity Assay

Seed cell suspensions (100 µL/well) in a 96-well plate.
Pre-incubate the plate for 24 h in an incubator at 37 °C and 5% CO₂.
Add 10 µL of test compound at various concentrations to the respective wells.
Incubate the plate for the desired experimental duration.
Add 10 µL of CCK-8 reagent to each well, avoiding bubble formation.
(Optional) If the test compound possesses inherent oxidative or reductive properties, the culture medium should be replaced with fresh, drug-free medium before adding CCK-8 to prevent artifactual results. For this, aspirate the existing medium, wash the wells twice with PBS or fresh medium, and then add 100 µL of new medium before the CCK-8 addition.
Incubate the plate for 2 h and measure the absorbance at 450 nm.
(Optional) If the absorbance cannot be measured immediately, the reaction may be stabilized by adding 10 µL of 0.1 M HCl or 1% (w/v) SDS solution to each well. The plate should then be covered and stored in the dark at room temperature; the absorbance values remain stable for up to 24 h under these conditions.

Cell Viability Calculation

Cell viability is expressed as a percentage and calculated using the following formula:

Cell Viability (%) = [A (treated) − A (blank)] / [A (untreated) − A (blank)] × 100

Where:

A (treated) is the absorbance in the well containing cells, CCK-8, and the test compound.
A (blank) is the absorbance in the well containing medium and CCK-8 only (no cells).
A (untreated) is the absorbance in the well containing cells and CCK-8 only (no test compound).

This calculation yields a value representing either cell proliferative activity or cytotoxic viability.

Notes and Technical Considerations

The CCK-8 assay is based on a dehydrogenase-catalyzed reduction reaction. Consequently, reducing agents and antioxidants present in the sample may confound the results and must be removed before analysis.
Preliminary experiments are strongly recommended to optimize key parameters, such as the density of seeded cells and the incubation period following CCK-8 addition.
Leukocytes and other non-adherent cell types may require extended culture times for adequate signal development.
For adherent cells in a standard 96-well plate, a minimum seeding density of 1,000 cells per well (in 100 µL medium) is advised. Due to lower assay sensitivity for leukocytes, a minimum of 2,500 cells per well is recommended.
When using different plate formats (e. g., 24-well or 6-well), scale the cell number and the volume of CCK-8 reagent proportionally, maintaining the CCK-8 volume at 10% of the total medium volume in the well.
Although the optimal absorbance maximum is 450 nm, filters within the range of 430–490 nm are acceptable, albeit with reduced sensitivity.
The background absorbance contributed by Phenol Red in the culture medium is automatically accounted for and subtracted during the calculation step via the blank well controls.
The presence of air bubbles in wells significantly alters absorbance measurements and must be removed before reading the plate.
Standard personal protective equipment (PPE), including safety glasses, gloves, and a laboratory coat, must be worn throughout this procedure.

Storage Conditions

Store at 0–5 °C. Transport: Up to 21 days at temperatures up to 25 °C.
CCK-8 is stable for one year when stored at 0–5 °C, protected from light.
For more extended storage, freeze and store at −20 °C.
Avoid repeated thawing and freezing, as this increases background levels, which interfere with assay results.
Expiration Date: 12 months from date of shipment.