Cck 8 method

Manufactured by Dojindo Laboratories

Sourced in Japan

The CCK-8 method is a colorimetric assay used to measure the viability and proliferation of cells. It utilizes a water-soluble tetrazolium salt, which is reduced by metabolically active cells to produce a colored formazan dye. The intensity of the color produced is directly proportional to the number of living cells, allowing for a quantitative assessment of cell growth and cytotoxicity.

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Lab products found in correlation

Apoptosis detection kit 1, by BD (2 mentions) C6 flow cytometer, by BD (2 mentions) Flx800 fluorescent microplate reader, by BioTeke (2 mentions) Microplate reader, by Thermo Fisher Scientific (2 mentions) Facscalibur, by BD (1 mentions) Rnase a, by Beyotime (1 mentions) Propidium iodide, by Beyotime (1 mentions) Varioskan flash, by Thermo Fisher Scientific (1 mentions) Matrigel coated 96 well plate, by Corning (1 mentions) Spectra mr, by Dynex (1 mentions) Concanavalin a (cona), by Merck Group (1 mentions) 37 c incubator, by Esco Lifesciences (1 mentions) U 2900 spectrophotometer, by Hitachi (1 mentions)

19 protocols using cck 8 method

Cell Proliferation and Cell Cycle Analysis

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For the evaluation of cell proliferation ability, the proliferation index of each group was determined using the CCK-8 method (Dojindo) according to the manufacturer’s instructions. In brief, 10 μL of CCK-8 solution was added into each well (containing 100 μL of medium), and further cultured for 1 h to 2 h at 37°C. The absorbance of each group at 450 nm was detected (n = 4) and it was directly proportional to the number of living cells. In our study, the proliferation index = the absorbance of experimental group—the absorbance of blank group, was used to measure cell proliferation ability.
To analyze cell cycle, the cells of each group were washed twice with PBS and harvested by trypsin. Then, the cells were fixed with 70% ethanol at 4°C for 12 hours. The fixed cells were washed with ice-cold PBS and stained with propidium iodide (Beyotime) in the presence of RNase A (Beyotime) for 30min as the manufacturer’s instructions. Finally, the cell cycle was analyzed using FACScalibur (BD Bioscience), and the software “FlowJo 7.6.1” was used to analyze the cell cycle results.

Liu P., Cai J., Dong D., Chen Y., Liu X., Wang Y, & Zhou Y. (2015). Effects of SOX2 on Proliferation, Migration and Adhesion of Human Dental Pulp Stem Cells. PLoS ONE, 10(10), e0141346.

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Assessing Cultured Neuron Viability

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The cultured hippocampal neurons were plated in 96-well cell culture clusters. The cell viability was assessed by CCK-8 method according to the manufacturer’s protocol (Dojindo Molecular Technology, MD, USA). Briefly, tetrazolium salt-8 (WST-8) solution was added to cell culture (10 μL/each well), followed by incubation at 37 °C for 2 h. The absorbance (optical density, OD, USA) was measured by spectrophotometry at 450 nm with an ELx-800 microplate reader (Bio-TekInc., Winooski, VT, USA). The cell viability was expressed as a percentage of control value.

Ding F., Bai Y., Cheng Q., Yu S., Cheng M., Wu Y., Zhang X., Liang X, & Gu X. (2021). Bidentatide, a Novel Plant Peptide Derived from Achyranthes bidentata Blume: Isolation, Characterization, and Neuroprotection through Inhibition of NR2B-Containing NMDA Receptors. International Journal of Molecular Sciences, 22(15), 7977.

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Cell Proliferation and Apoptosis Assay

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Cell proliferation assay was performed by CCK8 method (DOJINDO, Japan). Briefly, the about 5000 miR-137 mimic transfected cells and scramble cells were respectively seeded into 96-well plates and cultured. Proliferation rates were determined at 0, 12, 24, 48, 72, 96 hours after transfection by adding 10µl CCK8 regent and tested according to the manuscripts. The apoptosis assays were tested in HGC-27 and SGC-7901 cells lines with or without miR-137 overexpression by Apoptosis Detection kit I (BD Biosciences, USA) and C6 Flow Cytometer (USA).

Wu L., Chen J., Ding C., Wei S., Zhu Y., Yang W., Zhang X., Wei X, & Han D. (2015). MicroRNA-137 Contributes to Dampened Tumorigenesis in Human Gastric Cancer by Targeting AKT2. PLoS ONE, 10(6), e0130124.

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Cell Proliferation and Apoptosis Assays in Lung Cancer

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Cell proliferation assay was performed by the CCK8 method (DOJINDO, Japan). Briefly, approximately 5000 miR-217 mimic-transfected cells and scramble cells were seeded into 96-well plates and then cultured. Proliferation rates were determined at 0, 12, 24, 48, 72 and 96 h after transfection by adding 10 μl of CCK8 reagent.
Apoptosis assays were tested in SPC-A-1and A549 cell lines with or without miR-217 overexpression using Apoptosis Detection kit I (BD Biosciences, USA) and C6 Flow Cytometer (USA).

Guo J., Feng Z., Huang Z., Wang H, & Lu W. (2014). MicroRNA-217 Functions as a Tumour Suppressor Gene and Correlates with Cell Resistance to Cisplatin in Lung Cancer. Molecules and Cells, 37(9), 664-671.

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Cell Adhesion Assay via CCK-8

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Cell adhesion ability was examined by CCK-8 method (Dojindo). Briefly, 4×10⁴ cells suspended in 100 μL complete medium were seeded into each well of a matrigel-coated 96-well plate (Corning). After adhesion for 5 min, the floating cells were gently washed off with PBS.10 μL CCK-8 reagent was then added to each well of the plate and incubated for 2 hours at 37°C with 5%CO₂. The assay was performed in triplicate. The absorbance was measured at a wavelength of 450 nm by a Varioskan Flash (Thermo Scientific).

Dong X., Jiang Y., Liu J., Liu Z., Gao T., An G, & Wen T. (2018). T-Synthase Deficiency Enhances Oncogenic Features in Human Colorectal Cancer Cells via Activation of Epithelial-Mesenchymal Transition. BioMed Research International, 2018, 9532389.

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Splenocyte T Cell Cytokine and Proliferation Assay

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Splenocytes were cultured in complete medium stimulated by ConA (5 mg/L, Sigma) for 48 h. Cell-free supernatant fractions were collected and stored at -80°C until cytokines analysis by ELISA (ExCell Biology Inc., Shanghai, China). T cell differentiation was represented by Th1 cytokine secretion of IFNγ and Th2 cytokine secretion of IL-4. T cell function was represented by cell proliferation using a CCK-8 method (Dojindo Molecular Technologies Inc., Shanghai, China) in a multiplate spectrophotometer (Spectra MR; Dynex, Richfield, MN, USA) and IL-2 secretion by ELISA (ExCell Biology Inc.).

Ji X.J., Hao J.W., Li G.L., Dong N., Wang X.Q., Zhou M., Zhang Q.H, & Yao Y.M. (2019). Exendin-4 Exacerbates Burn-Induced Morbidity in Mice by Activation of the Sympathetic Nervous System. Mediators of Inflammation, 2019, 2750528.

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Assessing Ovarian Cell Viability via CCK-8

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The CCK-8 method (Dojindo Laboratories, Japan) was used to assess the relative viability of ovarian GCs. GCs were seeded in 96-well plates at a density of 1 x 10⁴ cells/well. After 24 h of treatment with 2,5-HD, 10 μL CCK-8 solution was added to each well and incubated in a 37 °C incubator (ESCO, Singapore) for 2 h. Then, the absorbance values were measured at a wavelength of 450 nm using an enzyme-labeled instrument. Cell viability was calculated with the following formula: (OD value - blank)/(OD value - blank) × 100%.

Sun Y., Chen H., Chen S., Xu X., Zhang W, & Li Y. (2023). The Hippo signaling pathway contributes to the 2,5-Hexadion-induced apoptosis of ovarian granulosa cells. Journal of Ovarian Research, 16, 161.

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Cell Proliferation Assay Using CCK-8

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To evaluate cell proliferation ability, the proliferation index was measured in different groups using CCK‐8 method (Dojindo, Japan) as the references.19, 20, 21 Briefly, 20 μL of the CCK‐8 solution was added into different wells, which contained 200 μL of medium, and was further incubated at 37°C for 4 hours. The absorbances (Abs) at 450 nm were detected respectively (n = 3). The wells containing only RPMI‐1640 medium were used as the blank group. Herein, the proliferation index = Abs of the experimental group—Abs of the blank group, was used to evaluate cell proliferation ability.

Zhao L., Chen X., Feng Y., Wang G., Nawaz I., Hu L, & Liu P. (2019). COX7A1 suppresses the viability of human non‐small cell lung cancer cells via regulating autophagy. Cancer Medicine, 8(18), 7762-7773.

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Cell Proliferation Assay by CCK-8

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Cell proliferation was detected by the commercial CCK-8 method (Dojindo Molecular Technologies, Inc., Kumamoto, Japan), according to the manufacturer's protocol. Shortly, cells were seeded into 96-well cell culture cluster plates at a concentration of 2×10⁴ cells/well in volumes of 100 µl, and cultured overnight. CCK-8 reagent was added to a subset of wells containing cells under different treatments, and incubated for 2 h at 37°C. The absorbance was next quantified at 450 nm with an automated plate reader.

GUAN C., ZHANG J., ZHANG J., SHI H, & NI R. (2016). Enhanced expression of early mitotic inhibitor-1 predicts a poor prognosis in esophageal squamous cell carcinoma patients. Oncology Letters, 12(1), 114-120.

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miR-29a Modulates Cell Proliferation

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A172 or U251 cells were transfected with miR-29a mimics or inhibitors and control oligoes for 24h. Then the cells were seeded into 96-well plates with a density of 2×10³ cells/well and maintained overnight. On days 1-4, cell proliferation was measured by CCK-8 method (Dojindo, Kumamoto, Japan) by absorbance at 450nm. Three independent experiments were performed in quadruplicate.

Liu Y., Duan N, & Duan S. (2018). MiR-29a Inhibits Glioma Tumorigenesis through a Negative Feedback Loop of TRAF4/Akt Signaling. BioMed Research International, 2018, 2461363.

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