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Short answer
The CCK-8 assay (Cell Counting Kit-8) is a colorimetric cell viability and proliferation assay. Its reagent contains WST-8, a water-soluble tetrazolium salt that cellular dehydrogenases reduce to an orange formazan dye. The dye is read as absorbance, usually at 450 nm, and the signal tracks metabolically active cells rather than counting them directly.
Definition and scope
CCK-8 is the trade name of Dojindo’s one-bottle WST-8 reagent, and several other vendors sell kits under the same or similar names. According to the manufacturer’s technical manual (CK04, revised 12 February 2020), WST-8 produces a water-soluble formazan dye on reduction in the presence of an electron mediator. The reagent is a one-bottle, nonradioactive solution that needs no premixing, and the orange product dissolves directly in the culture medium. The manufacturer states that the amount of formazan is directly proportional to the number of living cells.
Because the product is soluble, there is no solubilisation step. That is the practical difference from the MTT assay, where an insoluble formazan has to be dissolved before reading.
How it is used in practice
The manufacturer’s manual describes a 96-well format in which cells are seeded in 100 µL of medium per well and 10 µL of CCK-8 reagent is added to each well, taking care to avoid bubbles because they interfere with the optical density reading. Plates are incubated for 1 to 4 hours and read at 450 nm. For cytotoxicity work the manual’s example seeds 5,000 cells per well, pre-incubates for 24 hours, adds the test substance, incubates for a chosen exposure time (6, 12, 24 or 48 hours in the example), and then applies the reagent as above; the example figure uses 650 nm as a reference wavelength.
If plates cannot be read straight away, the manual describes adding 10 µL of 1% (w/v) SDS or 0.1 M HCl, covering the plate and protecting it from light at room temperature; it states that no absorbance change should be observed for 24 hours. The manual also says filters between 430 and 490 nm can be used, with 450 nm giving the best sensitivity.
Working ranges stated by the manufacturer for a 96-well plate: at least 1,000 adherent cells per well (at least 2,500 for leukocytes), and a recommended maximum of 25,000 cells per well. For other plate formats the manual advises scaling volumes so the reagent stays at 10% of the total volume. This page summarises stated parameters only; follow your kit’s own instructions for use for the actual protocol.
CCK-8 compared with MTT, XTT and MTS
All of these assays rely on tetrazolium reduction, but they differ in the reading wavelength and in whether the formazan is soluble. The wavelengths below are the ones in the manufacturer’s comparison figure.
| Assay | Wavelength in the Dojindo comparison | Related CASRAI guide |
|---|---|---|
| CCK-8 (WST-8) | 450 nm | This page |
| XTT | 450 nm | XTT assay |
| MTS | 490 nm | – |
| MTT | 570 nm | MTT assay |
Dojindo states that CCK-8 correlates with [3H]-thymidine incorporation and that its detection sensitivity is higher than MTT, XTT, MTS or WST-1. These are manufacturer claims, as are the statements on a competing vendor’s product page (AAT Bioquest) that its CCK-8 kit targets total dehydrogenase rather than mitochondrial dehydrogenase activity and has lower cytotoxicity than XTT. Treat comparative superiority statements as vendor claims to test in your own cell system, not as settled fact.
Where it matters for labs and supply decisions
Check the insert, not a generic protocol
Two vendors’ published details already differ, which is why the kit insert governs:
| Item | Dojindo CCK-8 (CK04 manual) | AAT Bioquest CCK-8 (product page) |
|---|---|---|
| Absorbance maximum quoted | 450 nm | 460 nm |
| Storage | 0 to 5 °C, stable over one year protected from light; -20 °C for longer storage | Below -15 °C, minimal light |
| MTT reading in comparison | 570 nm | 562 nm |
| Status | Nonradioactive one-bottle solution | Research use only |
Dojindo lists kit sizes of 100, 500, 2,500, 5,000 and 10,000 tests, notes that repeated freeze-thaw raises background, and says the solution can be sterilised by 0.2 µm filtration if needed. The manufacturer’s manual lists a 96-well plate and a plate reader with a 450 nm filter, and AAT Bioquest recommends a clear-bottom plate; see the guide to the plate reader for the instrument side.
Reporting checklist for methods sections
- Cell type, seeding density per well and plate format.
- Reagent incubation time (the manual says it varies with cell type and number).
- Reading wavelength and any reference wavelength.
- Blank (medium plus reagent) and compound-only controls, because the manual warns that reducing agents can generate formazan on their own.
- Kit vendor and lot.
Common misunderstandings
The signal is dehydrogenase activity, not a cell count
The manual cautions that conditions or chemicals that affect dehydrogenase activity may cause a discrepancy between the actual viable cell number and the number the assay reports. A treatment that changes cell metabolism without changing cell number can therefore move the readout. Where this matters, many labs pair the assay with a different readout such as the ATP assay or a direct count with a hemocytometer.
It is not a stain and it is not a death assay
The manufacturer states that the reagent does not stain cells because the product is water-soluble, and that its low toxicity allows the same cells to be used afterwards for crystal violet, neutral red or DNA fluorometric assays. The assay reports activity of living cells; it does not label dead cells.
Background and optical effects
- Phenol red can stay in the medium, provided the blank is subtracted.
- If the suspension is turbid, the manual advises subtracting the optical density at 600 nm or higher.
- Leukocytes give weak coloration, so the manual suggests incubating up to 4 hours or using around 105 cells per well.
FAQ
What does CCK-8 stand for?
Cell Counting Kit-8, the name of Dojindo’s WST-8-based kit; other vendors sell kits under the same or similar names.
What wavelength is used to read a CCK-8 assay?
Dojindo’s manual specifies 450 nm, with filters from 430 to 490 nm usable. Another vendor quotes a maximum near 460 nm, so use the wavelength in the insert of the kit you bought.
How long should cells be incubated with the reagent?
The manual describes 1 to 4 hours and says the optimum depends on cell type and cell number.
Is CCK-8 the same as the MTT assay?
No. Both depend on tetrazolium reduction by cells, but CCK-8 forms a formazan that dissolves in the medium, so no solubilisation step is described, and the readings are taken at different wavelengths.
Sources
- Dojindo Molecular Technologies, Cell Counting Kit-8 technical manual CK04, revised 12 February 2020: dojindo.com/products/manual/CK04e.pdf (checked 29 October 2026).
- AAT Bioquest, Cell Counting Kit-8 (CCK-8) product page: aatbio.com/products/cell-counting-kit-8-cck-8 (checked 29 October 2026).
Vendor statements are attributed to the vendor. This page is general information for laboratory staff and does not replace the instructions supplied with a kit.
