Cell Counting Kit-8 (CCK-8) for cell proliferation and cytotoxicity assays

Biotium products are distributed only in Singapore and Thailand.

Cell Counting Kit-8 (CCK-8) allows very convenient assays by utilizing the highly water-soluble tetrazolium salt WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium,monosodium salt] produces a water-soluble formazan dye upon reduction in the presence of an electron carrier. Cell Counting Kit-8 is a one-bottle solution; no premixing of components is required. Cell Counting Kit- 8, being nonradioactive, allows sensitive colorimetric assays for the determination of the number of viable cells in cell proliferation and cytotoxicity assays.

All products from TargetMol are for Research Use Only. Not for Human or Veterinary or Therapeutic Use.

Assay Mechanism

WST-8 is reduced by dehydrogenases in cells to give a yellowcolored product (formazan), which is soluble in the tissue culture medium. The amount of the formazan dye generated by the activity of dehydrogenases in cells is directly proportional to the number of living cells. The detection sensitivity of CCK-8 is higher than other tetrazolium salts such as MTT, XTT, MTS or WST-1.

Properties	MTT	XTT	WST-1	CCK8
Solublity of formazan	–	+	+	+
Forms	Powder	2-bottle solution	1-bottle solution	1-bottle solution
Preparation	Dissolve before use	Mix before use	Ready to use	Ready to use
Sensitivity	+	++	++	+++
Detection Speed	+	++	++	+++
Wavelerngth	560~600nM	420~480nM	420~480nM	430~490nM
Toxicity	+	–	–	–
Stability	+	–	+	++
96-well plate compatibility	+	++	++	++
Convenience	+	++	++	+++

Applications

Cell proliferation determinations
Cell viability assays
Cytokine assays
Cytotoxicity assays

Note

Cell Counting Kit-8 (CCK-8) is ready-to-use solution. Mix the reagent to ensure a homogenous solution before use.
Pay attention to the edge effect of 96-well plate. It is suggested to discard the surrounding plate hole and add the same amount of PBS.
This product is for R&D use only, not for medical, household, or other uses.

Advantages

More Sensitive than MTT, MTS, or WST-1
No Toxicity to Cell
Simpler Steps, No organic solvents required
Stable One Bottle Solution, ready-to-use

Experiment Procedure

1. 100μL cell suspension was inoculated on 96-well plate and incubated in cell incubator (37°C, 5% CO2). 2. Take the cells out of the incubator ,add 1/10 volume of Cell Counting Kit-8 (CCK-8) directly to cells in culture medium. Mix thoroughly to achieve a homogenous solution by lightly tapping the outside of the plate several times while avoiding bubbles. For 96-well plate, add 10 µl Cell Counting Kit-8 (CCK-8) per 100 µl culture medium. 3. Incubate in a cell culture incubator for 1 to 4 hours at 37°C until the color turns orange. Over incubation will give false results. 4. Place the 96-well plate on the shaking table for about 1min before the reading of the micrometer to ensure the uniform color of orifice plate. 5. The 450nm light absorption value was read by an enzyme marker and cell activity was calculated. 6. Optional: Add 10 μl of 1 % SDS (dissolve 0.1 g SDS with PBS buffer to prepare 10 ml solution) directly to 100 μl of cells to stop the reaction. Signals can be read within 3 days without affecting the absorbance values.

Features

The Cell Counting Kit-8 (CCK-8) is stable for 2 years at 4°C with protection from light. For long term storage, store at -20°C and below.

Reference Publication

Ni, Haiwei et al.

MiR-375 reduces the stemness of gastric cancer cells through triggering ferroptosis

Stem cell research & therapy vol. 12,1 325.

DOI: 10.1186/s13287-021-02394-7

Article Snippet: “Cell viability was assessed using CCK-8 (Cell Counting Kit-8) assay (Target Mol, USA). Cells were seeded in a 96-well plate at a density of 3000 cells/well in 100 μL of culture medium in a 5% CO₂ incubator at 37 °C for 24 h.”

Zhu L, Tian Q, Jiang S, et al.

A Novel Ferroptosis-Related Gene Signature for Overall Survival Prediction in Patients With Breast Cancer

Front. Cell Dev. Biol. 9:670184

DOI: 10.3389/fcell.2021.670184

Article Snippet: “MDA-MB-231 and MCF-7 cells were then treated with erastin (T-1765, TargetMol) of 0, 10, 20, and 40 μM for 24 and 48 h. The Cell Counting Kit-8 (CCK-8) (TargetMol) reagent was then added, and the 96-well plate was continued to incubate for another 2 h.”

Tan, J., Zhong, M., Hu, Y. et al.

Ritanserin suppresses acute myeloid leukemia by inhibiting DGKα to downregulate phospholipase D and the Jak-Stat/MAPK pathway

Discov Onc 14, 118 (2023).

DOI: https://doi.org/10.1007/s12672-023-00737-9

Article Snippet: “AML cells (1 × 10⁴ per well) were seeded in 100 μl medium in 96-well plates. The cell viability was determined by cell counting kit -8(CCK-8, TargetMol) after 24, 48 and 72 h of treatment with DMSO or ritanserin with specific concentration.”

Fang, XL., Li, QJ., Lin, JY. et al.

Transcription factor ATMIN facilitates chemoresistance in nasopharyngeal carcinoma

Cell Death Dis 15, 112 (2024).

DOI: https://doi.org/10.1038/s41419-024-06496-x

Article Snippet: “800 HONE1 cells or 1000 SUNE1 cells per well were seeded into the 96-well plates. On the indicated days from day 1 to day 5, 10 μl Cell Counting Kit-8 (CCK-8) reagent (TargetMol) per well was added to the 96-well plates.”

Li, J., Sun, Y., Zhao, X. et al.

Radiation induces IRAK1 expression to promote radioresistance by suppressing autophagic cell death via decreasing the ubiquitination of PRDX1 in glioma cells

Cell Death Dis 14, 259 (2023).

DOI: https://doi.org/10.1038/s41419-023-05732-0

Article Snippet: “Three thousand glioma cells per well in the exponential growth phase were seeded into 96-well plates (100 μL/well). At the indicated time of 6, 24, 48, 72, and 96 h after seeding, 10 μL CCK-8 solution (TargetMol, Boston, MA, USA) was added to each well and incubated for another 2 h.”

Wu, LL., Jiang, WM., Liu, ZY. et al.

AMG-510 and cisplatin combination increases antitumor effect in lung adenocarcinoma with mutation of KRAS G12C: a preclinical and translational research

Discov Onc 14, 91 (2023)

DOI: https://doi.org/10.1007/s12672-023-00698-z

Article Snippet: “To determine the IC50 value of AMG-510 and Cisplatin in H23 and H358 cell lines, we used Cell Count Kit8 (CCK-8, Targetmol Co. Ltd., Shanghai, China) after 72 h of drug treatment.”