CCK-8 Cell Viability Assay Protocol

1. Principle

Cell Counting Kit-8 (CCK-8) is a WST-8–based cell viability reagent commonly used for cell proliferation and cytotoxicity evaluation. In the presence of an electron-mediating agent, the water-soluble tetrazolium salt WST-8 is reduced by intracellular mitochondrial dehydrogenases to an orange formazan product. The product is soluble in the culture medium, and its color intensity correlates with intracellular enzyme activity, indirectly reflecting cell number and viability. When cells proliferate well and are metabolically active, more formazan is produced and the absorbance at 450 nm (OD) increases; when cells are damaged or killed by drugs or other factors, dehydrogenase activity decreases, less formazan forms, and the OD value decreases. Thus, by comparing OD changes between treated and control groups, the CCK-8 assay quantitatively assesses the effects of compounds or conditions on cell proliferation and cytotoxicity.

2.Procedure

（1）Prepare and count cell suspension:

For suspension cells, centrifuge and collect cells in log phase; for adherent cells, detach with trypsin and collect by centrifugation. Resuspend in serum-containing medium, count with a hemocytometer, and dilute to 5×10³–5×10⁴ cells/mL as a single-cell suspension.

（2）Seeding:

 In a 96-well plate, seed 100 μL per well. Set up different concentration groups with 3–6 replicates each, and include blank wells and control wells.

（3）Treatment and incubation:

Preincubate the plate ~24 h in a CO₂ incubator (37℃, 5% CO₂). Once adherence is satisfactory, remove medium and add test articles (drugs/chemicals) at desired concentrations. Incubate 6–96 h.

（4）CCK-8 incubation:

Add 10 μL CCK-8 solution to each well. Gently shake the plate to mix (to avoid reagent sticking to the wall). Avoid bubbles during addition, as they affect OD readings. Incubate 1–4 h in the incubator.

（5）OD measurement:

Read absorbance at 450 nm with a microplate reader.

3.Data Analysis

（1）Cell viability (%) = [(OD_experimental − OD_blank)/(OD_control − OD_blank)] × 100%

（2）Inhibition rate (%) = [(OD_blank − OD_experimental)/(OD_blank − OD_control)] × 100%

4.FAQ

Q1: Why is there large OD variation among replicates within the same group?

A: Common causes include uneven seeding (insufficient mixing, sedimentation during dispensing), inconsistent cell status (non-adherent or partially detached wells), edge-well evaporation, and bubbles when adding CCK-8. Mix the suspension thoroughly before seeding, preferentially use central wells, gently tap or shake the plate after seeding to distribute cells evenly, add CCK-8 slowly along the wall to avoid bubbles, and keep ≥3–6 replicates to smooth random error.

Q2: Color development is too weak and OD is low—what to do?

A: First confirm adequate seeding density and that cells are in log-phase growth, then check if incubation with reagent was too short. If control OD remains low, increase seeding density or extend incubation, e.g., from 1–2 h to 3–4 h; for slower-metabolizing cells (e.g., hematopoietic lines), 5–6 h is common. If still low, check whether medium, serum, or CCK-8 have expired or were stored improperly.

Q3: Color is too strong and OD approaches or exceeds 2.0—can I use it?

A: Aim to keep control OD (blank-subtracted) between ~1–2 for better linearity and quantitation. OD >2 suggests over-dense cells or overlong incubation near saturation, which can distort viability/inhibition curves. Reduce seeding density or shorten CCK-8 incubation, and determine a fixed “optimal color-development time” via a small pilot with multiple time points.

Q4: The drug itself is colored or absorbs at 450 nm—what now?

A: Include a “compound blank”: wells with drug + medium + CCK-8 but no cells. Use this OD as background for the compound. Subtract the corresponding blank from both experimental and control wells in calculations. If the compound is very dark/strongly absorbs at 450 nm and subtraction does not resolve interference, consider an alternative assay or wavelength.

Q5: Is CCK-8 toxic to cells? Can I measure repeatedly on the same plate?

A: At recommended concentrations, CCK-8 has low toxicity for most cells over short incubations. However, because color development relies on mitochondrial enzymes, repeated additions or prolonged incubations can gradually alter cell status. Do not perform multiple rounds on the same cells; typically add once and read the endpoint. For time-course monitoring, prepare separate plates for each time point.

Q6: Any special notes for suspension cells?

A: Prevent uneven settling and accidental aspiration of cells. Thoroughly mix before dispensing and complete plate loading quickly; avoid moving the plate during incubation; when adding CCK-8 or handling supernatant, do not insert tips too deep to avoid aspirating cells. If possible, briefly spin the plate before incubation to gently sediment cells to the bottom (use minimal g-force to avoid perturbing cell state).

Aladdin: https://www.aladdinsci.com/