|Cell Counting Kit-8 (CCK-8) Catalog No.GK10001|
Sample solution is provided at 25 µL, 10mM.
- 1. Zhang, Leike, et al. "Calcium channel blocker amlodipine besylate is associated with reduced case fatality rate of COVID-19 patients with hypertension." medRxiv (2020).
- 2. Lv, X. J., et al. "Long noncoding RNA PCAT6 regulates cell growth and metastasis via Wnt/β-catenin pathway and is a prognosis marker in cervical cancer." European review for medical and pharmacological sciences 23.5 (2019): 1947-1956. PMID:30915737
- 3. Meng, Y., Bai, X., Huang, Y., He, L., Zhang, Z., Li, X., ... & Yang, X. (2019). Basic fibroblast growth factor signalling regulates cancer stem cells in lung cancer A549 cells. Journal of Pharmacy and Pharmacology, 71(9), 1412-1420. PMID:31282010
- 4. Feng, Q., Li, Q., Wen, H., Chen, J., Liang, M., Huang, H., ... & Cao, X. (2019). Injection and Self‐Assembly of Bioinspired Stem Cell‐Laden Gelatin/Hyaluronic Acid Hybrid Microgels Promote Cartilage Repair In Vivo. Advanced Functional Materials.
- 5. Song, Qiongtao, et al. Puerarin protects against iron overload-induced retinal injury through regulation of iron-handling proteins. Biomedicine & Pharmacotherapy 122 (2020): 109690.
- 6. Li, Ling, et al. "Baicalin relieves TNF‐α‐evoked injury in human aortic endothelial cells by up‐regulation of miR‐145." Phytotherapy Research.
- 7. Tian, Chao, et al. "Transient Receptor Potential Ankyrin 1 Contributes to Lysophosphatidylcholine-Induced Intracellular Calcium Regulation and THP-1-Derived Macrophage Activation." The Journal of Membrane Biology (2019): 1-13.
- 8. Mei, Yi, Dongyan Cai, and Xiaofeng Dai. "Modulating cancer stemness provides luminal A breast cancer cells with HER2 positive-like features." Journal of Cancer 11.5 (2020): 1162-1169.
- 9. Yang, Shengzhuang, et al. "The long noncoding RNA LINC00483 promotes lung adenocarcinoma progression by sponging miR-204-3p." Cellular & Molecular Biology Letters 24.1 (2019): 1-14.
- 10. Mao, Qing, et al. "LncRNA KLF3-AS1 in human mesenchymal stem cell-derived exosomes ameliorates pyroptosis of cardiomyocytes and myocardial infarction through miR-138-5p/Sirt1 axis." Stem Cell Research & Therapy 10.1 (2019): 1-14.
- 11. Cao, Qifeng, et al. "ARID1A upregulation predicts better survival in patients with urothelial bladder carcinoma." Journal of International Medical Research (2019): 0300060519895687. PMID:31891283
- 12. Shi, Liang, et al. "miR-222 targets VGLL4 to promote growth of tongue squamous cell carcinoma." Materials Express 10.1 (2020): 102-111.
- 13. Xin, Suping, and Xinhua Ye. "Oxalomalate regulates the apoptosis and insulin secretory capacity in streptozotocin‐induced pancreatic β‐cells." Drug Development Research (2020). PMID:31904108
- 14. Zhou, Bing, et al. "Effect of miR-744 on Ameliorating Heart Allograft Rejection in BALB/c Mice Via Regulation of TNFRSF4 Expression in Regulatory T Cells." Transplantation Proceedings. Elsevier, 2020. PMID:31928781
- 15. Dou, Wenxue, et al. "Design and construction of a microporous CO 3 2--containing HA/β-TCP biphasic ceramic as a novel bone graft material." Materials Research Express (2020).
- 16. Yu, Jifeng, et al. "SPOP accelerates acute myeloid leukemia initiation and development through miR-183-mediated METAP2 inhibition."
- 17. Yu, Chaosheng, et al. "Pin2 telomeric repeat factor 1-interacting telomerase inhibitor 1 (PinX1) inhibits nasopharyngeal cancer cell stemness: implication for cancer progression and therapeutic targeting." Journal of Experimental & Clinical Cancer Research 39.1 (2020): 31.
- 18. Ye, Shuangyan et al. "Effect of the chemoprotectant tempol on anti-tumor activity of cisplatin" Journal of Southern Medical University 39.8 (2019): 883.
- 19. Xu, Guangru, et al. "Long non‐coding RNA POU6F2‐AS2 promotes cell proliferation and drug resistance in colon cancer by regulating miR‐377/BRD4." Journal of Cellular and Molecular Medicine (2020).
- 20. Zhang, Junjun, et al. "MALAT1 inhibits the Wnt/β-catenin signaling pathway in colon cancer cells and affects cell proliferation and apoptosis." Bosnian journal of basic medical sciences (2020).
- 21. Xie, Yingjie, et al. "Jianpi Huayu Decoction attenuates the immunosuppressive status of H22 hepatocellular carcinoma-bearing mice: by targeting Myeloid-derived suppressor cells." Frontiers in Pharmacology 11 (2020).
- 22. Yao, Xue, et al. "Nanoparticle‐Mediated Intracellular Protection of Natural Killer Cells Avoids Cryoinjury and Retains Potent Antitumor Functions." Advanced Science: 1902938.
- 23. Shen, Jingang, Xianbao Lv, and Lei Zhang. "GRHL2 Acts as an Anti-Oncogene in Bladder Cancer by Regulating ZEB1 in Epithelial-Mesenchymal Transition (EMT) Process." OncoTargets and therapy 13 (2020): 2511.
- 24. Huo, Xiu-zhu, et al. "Studies on the effect of a Fupenzi glycoprotein on the fibrillation of bovine serum albumin and its antioxidant activity." Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2020): 118387.
- 25. Pan, Zhenyu, et al. "Identification of Cerebrospinal Fluid MicroRNAs Associated With Leptomeningeal Metastasis From Lung Adenocarcinoma." Frontiers in Oncology 10 (2020).
- 26. Li, Yanting, et al. "MicroRNA-26b-3p inhibits human trophoblast cell proliferation, invasion and resistance to apoptosis via targeting SHBG." Journal of King Saud University-Science (2020).
- 27. Zhang, Qiao, et al. "Anti-colitic effects of Physalin B on dextran sodium sulfate-induced BALB/c mice by suppressing multiple inflammatory signaling pathways." Journal of Ethnopharmacology (2020): 112956.
- 28. Huang, Qingxia, et al. "Compound K inhibits autophagy-mediated apoptosis induced by oxygen and glucose deprivation/reperfusion via regulating AMPK-mTOR pathway in neurons." Life Sciences (2020): 117793.
Purity & Documentation
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Cell Number Determination
1. Inoculate cell suspension (100 μL/well) in a 96-well plate. Pre-incubate the plate in a humidified incubator (e.g., at 37°C, 5% CO2).
2. Add 10 μL of the CCK8 solution to each well of the plate. Be careful not to introduce bubbles to the wells, since they interfere with the O.D. reading.
3. Incubate the plate for 1 - 4 hours in the incubator.
4. Measure the absorbance at 450 nm using a microplate reader.
Cell Proliferation and Cytotoxicity Assay
1. Seed cells in a 96-well plate at a density of 103-104 cells/well in 100 μL of culture medium with or without compounds to be tested. Culture the cells in a CO2 incubator at 37°C for 24 hours.
2. Add 10 μL of various concentrations of substances to be tested to the plate.
3. Incubate the plate for an appropriate length of time (e.g., 6, 12, 24 or 48 hours) in the incubator.
4. Add 10 μL of CCK8 solution to each well of the plate using a repeating pipettor. Be careful not to introduce bubbles to the wells, since they interfere with the O.D. reading.
5. Incubate the plate for 1 - 4 hours in the incubator.
6. Before reading the plate, it is important to mix gently on an orbital shaker for 1 minute to ensure homogeneous distribution of color.
7. Measure the absorbance at 450 nm using a microplate reader.
There are several ways to do statistical analysis. You can choose to use O.D. values or cell numbers. We offer one of them.
Cell viability (%) = [(As-Ab) / (Ac-Ab)] × 100
Inhibition rate (%) = [(Ac-As) / (Ac-Ab)] × 100
As = absorbance of the experimental well (absorbance of cells, medium, CCK8 and wells of the test compound).
Ab = blank well absorbance (absorbance of wells containing medium and CCK8).
Ac = control well absorbance (absorbance of wells containing cells, medium and CCK8).
Making a standard curve
1. The cell counting plate counts the number of cells in the cell suspension.
2. Using the medium, the cell suspension is diluted to a concentration gradient, usually requiring 5-7 concentration gradients, several replicate wells per group. Then inoculate the cells. (Note the number of cells per well. If you are diluting the cell suspension in a tube, carefully mix the cells again before adding the wells to the plate. The volume of the cell suspension in each well should be the same.)
3. Incubate until the cells are adherent (usually 2-4 hours), then add 10 μl of CCK8 per 100 μl of medium. Incubation was continued for 1-4 hours, and the absorbance at 450 nm was measured with a microplate reader. Make a standard curve with the number of cells as the X-axis coordinate and the O.D. value as the Y-axis coordinate.
The number of cells of the sample to be tested can be determined based on the curve. A prerequisite for using this standard curve is that the culture conditions are the same.
1. Make sure the drug and CCK8 are evenly distributed in the medium.
2. The more cells proliferate, the darker the color; the stronger the cytotoxicity, the lighter the color.
3. For adherent cells, at least 1000 cells per well (100 μl medium). For leukocytes, at least 2500 cells per well (100 μl medium) are required due to their low sensitivity. The recommended 96-well plate has a maximum cell count of 25,000 per well. If the test is performed using a 24-well or 6-well plate, calculate the corresponding number of cells per well and adjust the volume of CCK8 to 10% of the total liquid volume per well.
4. Since the CCK8 assay is based on dehydrogenase activity in living cells, conditions or chemicals that affect dehydrogenase activity may result in a difference between the actual number of viable cells and the number of viable cells measured using CCK8.
5. WST-8 may react with a reducing agent to form WST-8 formazan. If a reducing agent (such as some antioxidants) is used, it will interfere with the test. If more reducing agent is present in the system to be tested, it is necessary to remove it.
6. After 2 hours of incubation, the background O.D. value is typically 0.1-0.2 units.
7. Be careful not to introduce air bubbles into the holes as they will interfere with the O.D. value.
8. If you want to sterilize the CCK8 solution, use a 0.2 μm membrane filter solution.
9. The incubation time will vary depending on the type and amount of cells in the well. Generally, leukocytes are less colored and may require longer incubation times (up to 4 hours) or large numbers of cells (~105 cells/well).
10. If there is high turbidity in the cell suspension, measure and subtract the O.D value of the sample at 600 nm or higher.
11. CCK8 cannot be used for cell staining.
12. The phenol red in the medium does not affect the experimental results. The absorbance of phenol red can be eliminated by subtracting the absorbance of the background in the blank hole during calculation, so it will not affect the detection.
13. The toxicity of CCK8 is very low. After the CCK8 assay is completed, the same cells can be used for other cell proliferation assays, such as crystal violet assay, neutral red assay or DNA fluorescence assay. (Unless the cells are extremely rare, it is not recommended.)
14. This kit can be used in E. coli but not in yeast cells.
15. Before reading the plate, you can mix gently on the shaker.
16. We recommend inoculation of cells in wells near the center of the plate. The medium in the outermost circle of holes is easily evaporated and can be filled with PBS, water or medium.
17. If you don't have a 450 nm filter. You can also use filters with absorbance between 430 and 490 nm, and 450 nm filters for optimum sensitivity.
18. Measure the absorbance at 450 nm. If you need to make a dual wavelength measurement, the absorbance at 650 nm can be determined as the reference wavelength.
19. The presence of metal ions in the drug may affect the sensitivity of CCK8. Lead chloride, iron chloride, and copper sulfate at a final concentration of 1 mM inhibit 5%, 15%, and 90% of the color reaction, reducing sensitivity. If the final concentration is 10 mM, it will be 100% inhibited.
Features & Properties
1) Cell proliferation determinations-the GlpBio Cell Counting Kit-8 (CCK-8) is water soluble, stable in culture, and non-toxic.
2) Cell viability assays-metabolic activity and dye generation changes in proportion to altered viability.
3) Cytokine assays-measure cytokine-induced proliferation. Cells can be recovered and expanded at the end of the study if desired.
4) Cytotoxicity assays-Cells death from cytotoxic chemicals has no effects on color development, only living cells convert the reagent into a colorimetric indicator. The reagent itself has negligible toxicity, and is generally safe for cells.
|Shipping||Ship with blue ice.|
|Storage Conditions||Stored at 4°C protecting from light, and is stable for up to 12 months. Stored at -20°C protecting from light, and is stable for up to 2 years.|
|Usage||For Research Use Only! Not For Use in Humans.|
|Solubility||Water soluble||Indissolvable||Water soluble||Indissolvable|
|Usage||No need to prepare||Prepare the solutions||Use it right after it was ready||Prepared beforehand|
|Need to redissolve or not||NO||Yes,by DMSO||No||Yes,by Tris-base solution|
Cell Counting Kit-8 (CCK-8) allows convenient assays using WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(
Cell Counting Kit-8 allows sensitive colorimetric assays for the determination of the number of viable cells in the proliferation and cytotoxicity assays. The detection sensitivity is higher than any other tetrazolium salts such as MTT, XTT or MTS.
Figure 1: Working mechanisms of Cell Counting Kit-8 (CCK-8).