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| Product Name | Cell Counting Kit-8 (CCK-8) |
| Catalog No. | RESK-0137 |
| Description | The Cell Counting Kit-8 (CCK-8) is a rapid, highly sensitive kit based on WST-8 [chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] and is widely used for the detection of cell proliferation and cytotoxicity. WST-8 is an improved tetrazolium salt relative to MTT: in the presence of an electron coupling reagent, it is reduced by dehydrogenases in mitochondria to a highly water-soluble orange-yellow formazan product. The color intensity is directly proportional to cell proliferation and inversely proportional to cytotoxicity. The OD value measured at 450 nm with a microplate reader indirectly reflects the number of viable cells. The CCK-8 assay is widely applied in drug screening, cell proliferation assays, cytotoxicity assays, tumor drug-sensitivity testing, and the activity detection of biological factors. |
| Intended Use | Rapid and highly sensitive detection of cell proliferation and cytotoxicity Drug screening and high-throughput sample analysis Tumor drug-sensitivity testing Activity detection of biological factors Detection of E. coli (not suitable for yeast cells) |
| Principle / Technology | In the presence of an electron coupling reagent, WST-8 is reduced by mitochondrial dehydrogenases in living cells to a highly water-soluble orange-yellow formazan product. The amount of formazan (color intensity) is directly proportional to cell proliferation and inversely proportional to cytotoxicity, and is quantified by measuring the OD at 450 nm with a microplate reader. Unlike MTT, the formazan product is water-soluble and requires no organic solvent for dissolution. The whole color reaction takes place in the culture system and does not stain living cells. |
| Detection Method | Colorimetric (absorbance) assay; measure the OD at 450 nm with a microplate reader. |
| Sample Type | Cultured adherent or suspension cells. Also applicable to E. coli: add 10 uL of CCK-8 solution to 100 uL of E. coli culture and incubate for 1-4 h or overnight. Not suitable for yeast cells. |
| Performance Range / Specifications | Simple operation: supplied as a ready-to-use liquid; the water-soluble formazan product requires no organic solvent High sensitivity with a wide linear range and good reproducibility Very low cytotoxicity, with no impact on subsequent experiments Good stability: phenol red and serum in the culture medium do not affect the detection Broad applicability: suitable for large-scale, high-throughput sample detection |
| Sensitivity / LOD | High sensitivity with a wide linear range; viable cell number is quantified indirectly via the OD450 value. |
| Reaction Conditions / Protocol | Pre-culture of cells is recommended to keep intracellular dehydrogenases stable; if pre-culture is omitted, keep the standard curve and detection conditions consistent. Add 10 uL of CCK-8 solution per 100 uL of cell culture and incubate for 1-4 h, then measure the OD at 450 nm. Medium replacement before adding CCK-8 is generally unnecessary, but is required when the medium contains redox-active substances. If the OD value is too high, shorten the incubation time after adding CCK-8 (e.g., from 2 h to 1 h) or reduce the cell number; if it is too low, increase the cell number or prolong the reaction time. |
| Components / Formulation | Ready-to-use liquid reagent containing WST-8 and an electron coupling reagent. |
| Storage Conditions | Store at -25 to -15 °C, protected from light and dry. For frequent use, store at 4 °C and avoid repeated freeze-thaw cycles. The normal color of the reagent is pink; a marked deviation in color may indicate a change in quality. |
| Shelf Life | 2 years at -25 to -15 °C (protected from light); 1 year at 4 °C. |
| Package Specifications | 100 T 500 T 1000 T 3× 1000 T 10× 1000 T |
| Product Form | Ready-to-use liquid reagent (normal color: pink) |
| Shipping Conditions | Shipped with ice packs. |
| Expiration Date / Stability | Highly stable: valid for 2 years at -20 °C protected from light and 1 year at 4 °C. Avoid repeated freeze-thaw cycles; a marked change from the normal pink color may indicate quality deterioration. |
| Compatibility | Because of its very low cytotoxicity, the same cells can be used after CCK-8 detection for other proliferation assays such as crystal violet staining, neutral red staining, or DNA fluorescent staining. WST-8 and its formazan are highly water-soluble; replacing with fresh medium after the reaction removes exogenous components. |
| Recommended Buffer System | No special buffer required; the reagent is added directly to the cell culture medium. Phenol red background absorbance can be eliminated by subtracting the absorbance of blank wells. |
| Application Notes / Precautions | Reducing substances in the culture system can reduce WST-8 and increase the OD value; oxidizing substances can inhibit the reduction and decrease the OD value. LDH released from apoptotic or dead cells persists in the culture supernatant and can elevate the OD value, so cell viability and OD value are not fully equivalent; replace with fresh medium when adding CCK-8 for accurate cytotoxicity results. A high blank control (medium + CCK-8) value may be caused by redox-active substances in the medium. Cell viability and OD value are not fully equivalent; ensure consistent detection conditions when comparing samples. |
For research use only, not for clinical use.
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