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SKU AR1160
Product nameCell Counting Kit-8
Pack size 5 mL (for 500 assays, 10μL per well)

Product Overview

Form Liquid
Size 5 mL (for 500 assays, 10μL per well)
Concentration 6mg/ml
Buffer 9% NaCl
Storage Upon receipt store at -4°C, protect from light.
Note: Repeated thawing and freezing causes an increase in the background, which interferes with the assay.
Stability When stored as directed, product should be stable for one year. Store it at -20˚C for longer storage
Sensitivity CCK-8 is the highest sensitive dye for the cell based assay.
Equivalent Dojindo Molecular Technologies (Product No. CK04);
Millipore Sigma (Product No. 96992)

Introduction

Cell Counting Kit-8 (CCK-8) allows very convenient assays by utilizing 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 cytotoxicity assays. WST-8 is reduced by dehydrogenases in cells to give an orange colored 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 cell proliferation assay using CCK-8 correlates well with the [(3)H] thymidine incorporation assay. And the CCK-8 assay can also be substituted for the [(3)H]thymidine incorporation assay. The detection sensitivity of CCK-8 is higher than other tetrazolium salts such as MTT, XTT, MTS or WST-1.

Advantages

1. One bottle, ready to use solution.

2. No organic solvents or isotopes required

3. No harvesting, no washing and no solubilization steps.

4. No Toxicity to Cell

5. More sensitive than MTT, XTT, MTS or WST-1

Precautions

1. Run pilot test to determine the optimal cell number and incubation time.

2. Since the CCK-8 assay is based on the dehydrogenase activity detection in viable cells, conditions or chemicals that affect dehydrogenase activity in viable cells may cause discrepancy between the actual viable cell number and the cell number determined using the CCK-8 assay.

3. WST-8 may react with reducing agents to generate WST-8 formazan. Please check the background O.D. if reducing agents are used in cytotoxicity assays or cell proliferation assays.

4. Be careful not to introduce bubbles to the wells, since they interfere with the O.D. reading.

5. Phenol red containing culture media can be used with this kit for cell viability assays.

6. Membrane filtration is recommended for the sterilization of the CCK-8 solution, if necessary.

7.The incubation time varies by the type and number of cells in a well. Generally, leukocytes give weak coloration, thus a long incubation time (up to 4 hours) or a large number of cells (~105 cells/well) may be necessary.

8. Since the cytotoxicity of this kit is very low, further color development is possible after reading the absorbance.

9.Neutral red or crystal violet can be used after the CCK-8 assay.

10.Measure the reference wavelength at 600 nm or higher if there is a high turbidity in the cell suspension.

Additional Materials Required

10μL and 100-200μL Multichannel Pipettes

Plate reader (450 nm filter)

96-well plate

CO2 incubator

General Protocol

Cell Number Determination

1. Inoculate cell suspension (100μL/well) in a 96-well plate. Pre-incubate the plate in a humidified incubator (e.g., at 37 ˚C, 5% CO2).

2. Add 10μL of the CCK-8 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. To measure the absorbance later, add 10μL of 1% w/v SDS or 0.1M HCl to each well, cover the plate and store it with protection from light at room temperature. No absorbance change should be observed for 24 hours.

Cell proliferation and Cytotoxicity Assay

1. Dispense 100μL of cell suspension (5000 cells/well) in a 96- well plate. Pre-incubate the plate for 24 hours in a humidified incubator (e.g., at 37 ˚C, 5% CO2).

2. Add 10μL 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 10μL of CCK-8 solution to each well of the plate. 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. Measure the absorbance at 450 nm using a microplate reader. To measure the absorbance later, add 10μL of 1% w/v SDS or 0.1M HCl to each well, cover the plate and store it with protection from light at room temperature. No absorbance change should be observed for 24 hours.

Frequently Asked Questions

1. How many cells should there be in a well?

For adhesive cells, at least 1000 cells are necessary per well (100μL medium). For leukocytes, at least 2500 cells are necessary per well (100μL medium) because of low sensitivity. The recommended maximum number of cells per well for the 96-well plate is 25000. If a 24 well or 6 well plate is used for this assay, please calculate the number of cells per well accordingly, and adjust the volume of the CCK-8 solution in a well to 10% of the total volume.

2. Does CCK-8 stain viable cells?

No. Since WST-8 and its formazan dye are highly water-soluble, CCK-8 cannot be utilized for cell staining purpose.

3. Does phenol red affect the assay?

No. The absorption value of phenol red in a culture medium can be removed by subtracting the absorption value of a blank solution from the absorption value of each well. Therefore, a medium containing phenol red is usable for the CCK-8 assay.

4. Is CCK-8 toxic to cells?

Since the toxicity of CCK-8 is so low, the same cells can be used for other cell proliferation assays such as the crystal violet assay, neutral red assay or DNA fluorometric assay after the CCK-8 assay is completed.

5. I do not have a 450 nm filter. What other filters can I use?

You can use filters with the absorbance between 430 and 490 nm, even though 450 nm filter gives the best sensitivity.

Cell Counting Kit-8 (CCK-8) Images

Click the images to enlarge.

Cell Counting Kit-8 (CCK-8)
Figure 1. Absorption spectrum of WST-8 formazan
Figure 1 shows the absorption spectrum of WST-8 formazan. Since the absorbance at 460 nm is proportional to the number of viable cells in the medium, the viable cell number can be determined using the absorbance of a previously prepared calibration curve.
Cell Counting Kit-8 (CCK-8)
Figure 2. Cell proliferation assay using CCK-8
Culture medium: MEM, 10% FBS
Incubation: 37 ˚C, 5% CO2, 2 hours
Deteactin: 450nm
Cell Counting Kit-8 (CCK-8)
Figure 3. Toxicological test of chemicals using CCK-8
Cell line: hela
Medium: DMEM, 10% FBS Chemicals: 200 μM Cisplatin (DDP)
Incubation: 37°C, 5% CO2, 2 hours
Cell Counting Kit-8 (CCK-8)
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Publications

CPNE1 is a target of miR-335-5p and plays an important role in the pathogenesis of non-small cell lung cancer
Expression of cold-inducible RNA-binding protein (CIRP) in renal cell carcinoma and the effect of CIRP downregulation cell proliferation and chemosensitivity to gemcitabine
C/EBP? enhances platinum resistance of ovarian cancer cells by reprogramming H3K79 methylation
Human nail stem cells are retained but hypofunctional during aging
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Clinical significance of LMO1 in gastric cancer tissue and its association with apoptosis of cancer cells
MiR-491-5p negatively regulates cell proliferation and motility by targeting PDGFRA in prostate cancer
Effects of vaspin on pancreatic ? cell secretion via PI3K/Akt and NF-?B signaling pathways
Association of protein kinase CK2 inhibition with cellular radiosensitivity of non-small cell lung cancer
Fibroblast growth factor 21 delayed endothelial replicative senescence and protected cells from H2O2-induced premature senescence through SIRT1
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Iguratimod prevents ovariectomy-induced bone loss and suppresses osteoclastogenesis via inhibition of peroxisome proliferator-activated receptor-?
Chikusetsu saponin IVa attenuates isoflurane-induced neurotoxicity and cognitive deficits via SIRT1/ERK1/2 in developmental rats
Inhibition of autophagy potentiates the proliferation inhibition activity of microRNA-7 in human hepatocellular carcinoma cells
Potential role of ZEB1 as a DNA repair regulator in colorectal cancer cells revealed by cancer-associated promoter profiling
Long non-coding RNA linc00673 regulated non-small cell lung cancer proliferation, migration, invasion and epithelial mesenchymal transition by sponging miR-150-5p
miR-34a mediates oxaliplatin resistance of colorectal cancer cells by inhibiting macroautophagy via transforming growth factor-?/Smad4 pathway
Overexpression of decorin promoted angiogenesis in diabetic cardiomyopathy via IGF1R-AKT-VEGF signaling
MicroRNA-205 targets SMAD4 in non-small cell lung cancer and promotes lung cancer cell growth in vitro and in vivo
CLASP2 is involved in the EMT and early progression after transurethral resection of the bladder tumor
CD73/NT5E is a target of miR-30a-5p and plays an important role in the pathogenesis of non-small cell lung cancer
Three-dimensional Printed Scaffolds with Gelatin and Platelets Enhance In vitro Preosteoblast Growth Behavior and the Sustained-release Effect of Growth Factors
Downregulation of ZEB2-AS1 decreased tumor growth and metastasis in hepatocellular carcinoma
THBS2 is a Potential Prognostic Biomarker in Colorectal Cancer
IL-13/STAT6 signaling plays a critical role in the epithelial-mesenchymal transition of colorectal cancer cells
Effects of cisplatin on the LSD1-mediated invasion and metastasis of prostate cancer cells
Actein Inhibits Cell Proliferation and Migration in Human Osteosarcoma
Upregulated ATF6 contributes to chronic intermittent hypoxia-afforded protection against myocardial ischemia/reperfusion injury
Downregulation of HIF-1? inhibits the proliferation and invasion of non-small cell lung cancer NCI-H157 cells
SENP?1 enhances hypoxia?induced proliferation of rat pulmonary artery smooth muscle cells by regulating hypoxia?inducible factor?1?
The Mycobacterium bovis BCG prime-Rv0577 DNA boost vaccination induces a durable Th1 immune response in mice
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Downregulation of caveolin-1 upregulates the expression of growth factors and regulators in co-culture of fibroblasts with cancer cells
Demethylation of miR-10b plays a suppressive role in ccRCC cells
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Single peptide ligand-functionalized uniform hollow mesoporous silica nanoparticles achieving dual-targeting drug delivery to tumor cells and angiogenic blood vessel cells
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Overexpression of Wip1 Is Associated with Biologic Behavior in Human Clear Cell Renal Cell Carcinoma
Hes-1 SUMOylation by protein inhibitor of activated STAT1 enhances the suppressing effect of Hes-1 on GADD45? expression to increase cell survival
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Mono-(2-Ethylhexyl) Phthalate Induces Injury in Human Umbilical Vein Endothelial Cells
Overexpression of SATB1 Is Associated with Biologic Behavior in Human Renal Cell Carcinoma
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miR-21, miR-17 and miR-19a induced by phosphatase of regenerating liver-3 promote the proliferation and metastasis of colon cancer
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Lai J, Chen F, Chen J, Ruan G, He M, Chen C, Tang J, Wang DW. Sci Rep. 2017 Mar 14;7:44473. doi: 10.1038/srep44473. Overexpression of decorin promoted angiogenesis in diabetic cardiomyopathy via IGF1R-AKT-VEGF signaling
Yang Y, Hou J, Shao M, Zhang W, Qi Y, E S, Wang S, Sui H, Meng D, Wang B, Wang M, Han Y, Cao Y, Huang X, Li Y, Zhang P, Wang W. Oncol Lett. 2017 Dec;14(6):7977-7985. doi: 10.3892/ol.2017.7236. Epub 2017 Oct 20. CXCL5 as an autocrine or paracrine cytokine is associated with proliferation and migration of hepatoblastoma HepG2 cells
Wu YX, Sun Y, Ye YP, Zhang P, Guo JC, Huang JM, Jing XZ, Xiang W, Yu SY, Guo FJ. Mol Med Rep. 2017 Dec;16(6):8200-8208. doi: 10.3892/mmr.2017.7648. Epub 2017 Sep 28. Iguratimod prevents ovariectomy-induced bone loss and suppresses osteoclastogenesis via inhibition of peroxisome proliferator-activated receptor-γ
Guo J, Li W, Wu Y, Jing X, Huang J, Zhang J, Xiang W, Ren R, Lv Z, Xiao J, Guo F. Front Pharmacol. 2017 Oct 4;8:693. doi: 10.3389/fphar.2017.00693. eCollection 2017. Meclizine Prevents Ovariectomy-Induced Bone Loss and Inhibits Osteoclastogenesis Partially by Upregulating PXR
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Zhu Y, Lu H, Zhang D, Li M, Sun X, Wan L, Yu D, Tian Y, Jin H, Lin A, Gao F, Lai M. Clin Epigenetics. 2018 Mar 2;10:30. doi: 10.1186/s13148-018-0458-3. eCollection 2018. Integrated analyses of multi-omics reveal global patterns of methylation and hydroxymethylation and screen the tumor suppressive roles of HADHB in colorectal cancer
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VOL. 73 No. 6 November/December 2016 ISSN 2353-5288
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Zhang J, Xiao Z, Lai D, Sun J, He C, Chu Z, Ye H, Chen S, Wang J. Br J Cancer. 2012 Jul 10;107(2):352-9. Doi: 10.1038/Bjc.2012.251. Epub 2012 Jun 7. Mir-21, Mir-17 And Mir-19A Induced By Phosphatase Of Regenerating Liver-3 Promote The Proliferatio...
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Yu G, Li H, Wang X, Wu T, Zhu J, Huang S, Wan Y, Tang J. Mol Cell Biochem. 2013 Aug;380(1-2):239-47. Doi: 10.1007/S11010-013-1679-6. Epub 2013 May 12. Microrna-19A Targets Tissue Factor To Inhibit Colon Cancer Cells Migration And Invasion.
Chiou Hy, Liu Sy, Lin Ch, Lee Eh. J Biomed Sci. 2014 Jun 4;21:53. Doi: 10.1186/1423-0127-21-53. Hes-1 Sumoylation By Protein Inhibitor Of Activated Stat1 Enhances The Suppressing Effect Of Hes-1 On Gadd45?? Expression To Increase Cell Survival.
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