How to Perform a Valid CCK-8 Cytotoxicity Assay

Views :

Update time : 2026-04-10

Commonly used methods for cytotoxicity testing include the MTT assay and the CCK-8 assay. Compared with the MTT assay, the CCK-8 assay is widely used due to its non-toxicity and high efficiency. This article introduces the procedure and practical tips for the CCK-8 assay.

Principle of the CCK-8 Assay

CCK-8, short for Cell Counting Kit-8, is a rapid and highly sensitive detection kit based on WST-8, used for measuring cell proliferation and cytotoxicity.

In the presence of an electron-coupling reagent, WST-8 can be reduced by mitochondrial dehydrogenase to an orange-yellow formazan product. The color intensity is directly proportional to the degree of cell proliferation and inversely proportional to the degree of cytotoxicity.

CCK-8 Assay Procedure

1. Prepare cell suspension

￮ For suspension cells: directly collect cells in the logarithmic growth phase by centrifugation.

￮ For adherent cells: digest with trypsin and collect by centrifugation.

Resuspend the collected cells in serum-containing medium, count with a hemocytometer, and dilute to a single-cell suspension of 5×10³–5×10⁴ cells/mL.

2. Seed cells into a 96-well plate

Add 100 μL of cell suspension per well. Set up different concentration groups of the test substance, with 3–6 replicate wells per group, and include blank and control groups.

3. Pre-incubate the plate

Place the culture plate in an incubator for approximately 24 h (37 °C, 5% CO₂).

4. Treat with test substance

Add test substances (drugs, chemicals, etc.) at various concentrations to each well, and incubate in the incubator for 6–96 h.

5. Add CCK-8 solution

Add 10 μL of CCK-8 solution to each well. Gently tap or swirl the plate to ensure even mixing (to avoid errors from reagent adhering to well walls). Avoid generating bubbles during pipetting, as they interfere with OD readings.

6. Incubate for color development

Return the plate to the incubator and incubate for 1–4 h.

7. Measure absorbance

Detect the absorbance (OD value) at 450 nm using a microplate reader.

Notes

1. Cell seeding density

For a standard 96-well plate:

￮ Minimum seeding density for adherent cells: 1,000 cells/well (100 μL medium)

￮ Minimum seeding density for leukocytes: 2,500 cells/well (100 μL medium)

For 24-well or 6-well plates, calculate the appropriate seeding density first, and add CCK-8 solution at 10% of the total medium volume per well.

2. Drug absorption correction

In cytotoxicity tests, consider drug absorbance. Add CCK-8 to drug-containing medium and measure absorbance at 450 nm as a blank control.

3. Edge effect avoidance

The outermost wells of the plate tend to dry out during incubation, causing volume inaccuracy and increased error. It is recommended to fill only with PBS and not use them for measurement.

4. Incubation time with test substance

The incubation period (6–96 h) depends on the nature of the test substance and cell sensitivity (e.g., 6, 12, 24, 48 h). Cytotoxicity can be evaluated at multiple time points.

5. Redox-active test substances

If the sample has oxidizing or reducing properties, replace with fresh medium before adding CCK-8 to eliminate interference. If the effect is negligible, medium replacement is unnecessary; simply subtract the blank absorbance of drug-containing medium.

6. Color development time

Normal color development takes 1–4 h. Measure OD every 30 minutes until the OD value ranges between 1 and 2, then use this fixed time for repeated assays.

7. Cell type-dependent color development

Formazan production varies by cell type. Insufficient color can be resolved by extending incubation time; blood cells often require 5–6 h.

8. Wavelength selection

For highly turbid cell suspensions, dual-wavelength measurement is recommended:

￮ Detection wavelength: 450–490 nm

￮ Reference wavelength: 600–650 nm (650 nm is preferred)

9. Delayed OD measurement

If OD readings cannot be taken immediately, add 10 μL of 0.1 M HCl or 1% (w/v) SDS to each well. Store protected from light at room temperature; OD values remain stable within 24 h.

10. CCK-8 reagent storage

Fresh CCK-8 is pink; aged reagent darkens and loses efficiency and should be discarded.

￮ 0–5 °C, protected from light: stable for at least 6 months

￮ −20 °C, protected from light: stable for 1 year

Common Issues

Poor repeatability between replicate wells

1. Bubbles introduced during seeding

Use fresh pipette tips, dispense cell suspension along the well wall, and avoid submerging the tip below the liquid surface. Existing bubbles can be popped with a heated 1 mL syringe needle.

2. Dislodged adherent cells during medium change

After cell attachment, carefully aspirate medium from the bottom along the well wall using the siphon effect of the pipette tip.

3. Incomplete removal of old medium

Ensure thorough aspiration of residual liquid before adding fresh medium.

4. Inaccurate CCK-8 dispensing

Prepare medium containing 10% CCK-8 and add 100 μL to each well via medium replacement to improve precision.