Cell counting kit 8 (cck8)

Manufactured by Nanjing Jiancheng

Sourced in China

The Cell Counting Kit-8 is a colorimetric assay for the determination of cell viability and cytotoxicity. It utilizes the highly water-soluble tetrazolium salt WST-8, which is reduced by dehydrogenases in viable cells to produce a yellow-colored formazan dye. The amount of the formazan dye generated is directly proportional to the number of living cells.

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Lab products found in correlation

Microplate reader, by Thermo Fisher Scientific (2 mentions) Automatic microplate reader, by Bio-Rad (1 mentions) Annexin 5 fitc pi, by Thermo Fisher Scientific (1 mentions) Anti β actin mab, by Cell Signaling Technology (1 mentions) Paraquat, by Merck Group (1 mentions) Mitotracker green fm, by Thermo Fisher Scientific (1 mentions) Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions) Hoechst 33342, by Merck Group (1 mentions) Annexin 5 fitc propidium iodide apoptosis detection kit, by Keygen Biotech (1 mentions) Mdivi 1, by Enzo Life Sciences (1 mentions) Alexa fluor 555 labeled secondary antibody, by Beyotime (1 mentions) Cck 8, by Thermo Fisher Scientific (1 mentions) Cck 8 reagent, by MultiSciences Biotech (1 mentions) Microplate reader, by Agilent Technologies (1 mentions)

11 protocols using cell counting kit 8 (cck8)

Artemisinin Effects on pMEC Viability and Apoptosis

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To test the cell viability, equal amounts of pMECs were seeded in 96-well plates at a density of 2 × 10⁴ cells/mL and cultured in DMEM/F12 containing 10% FBS. After cultured for 48 h, cells are washed three times with PBS, and then treated with different concentrations of artemisinin or LPS for different times. The cell counting kit - 8 (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was used to analyze cell viability according to the instruction. An automatic microplate reader (Bio-Rad Laboratories Inc., Foster City, CA) was used to measure the absorbance of all wells at a wavelength of 450 nm. Cell viability is the quantification of No. live cells and is expressed as a percentage of the control.
To test cell apoptosis, pMECs were pretreated with 0 or 20 μM of artemisinin for 12 h before being stimulated with 50 μg/mL LPS for 24 h. After treatment, all cells were trypsinized, washed 2~3 times with PBS, stained with Annexin V-FITC/PI (Invitrogen Inc., Carlsbad, CA, USA), and analyzed by flow cytometry, according to the instructions.

Zhang W., Xiong L., Chen J., Tian Z., Liu J., Chen F., Ren M., Guan W, & Zhang S. (2021). Artemisinin Protects Porcine Mammary Epithelial Cells against Lipopolysaccharide-Induced Inflammatory Injury by Regulating the NF-κB and MAPK Signaling Pathways. Animals : an Open Access Journal from MDPI, 11(6), 1528.

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Cell Viability Determination by CCK-8 Assay

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Cell viability was determined by the Cell Counting Kit-8 (CCK-8) assay (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China). Cells (3 × 10⁴ cells/well) were seeded into 96-well tissue culture plates. After succinate treatment, CCK-8 was used to stain the cells for about 2 h. The optical density was measured at 450 nm using a microplate spectrophotometer.

Li X., Mao M., Zhang Y., Yu K, & Zhu W. (2019). Succinate Modulates Intestinal Barrier Function and Inflammation Response in Pigs. Biomolecules, 9(9), 486.

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Paraquat-Induced Oxidative Stress Protocols

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Paraquat, 2′, 7′- dichlorodihydrofluorescein diacetate (DCFH-DA), Hoechst 33342 was purchased from Sigma-Aldrich (St Louis, MO, USA). Dulbecco's Modified Eagle Medium (DMEM) used for AT-II cell culture was obtained from Gibco (Carlsbad, CA, USA). Cell counting kit-8 was from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Annexin V-FITC/propidium iodide (PI) apoptosis detection kit was obtained from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). Mitochondrial division inhibitor 1 (Mdivi-1) and anti-Fis1 mAb was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Ascorbic Acid was purchased from Biogot Technology (Nanjing, China). MitoTracker Green FM were provided by Molecular probes (Eugene, OR, USA). Anti-Drp1 mAb, anti-Opa1 mAb, anti-Fis1 mAb, anti-Mfn2 mAb and anti-β-actin mAb used in this study were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Cyt C antibody, anti-Cox IV antibody and Alexa Fluor 555 labeled secondary antibody were provided by Beyotime Co., Ltd. (Shanghai, China).

Zhao G., Cao K., Xu C., Sun A., Lu W., Zheng Y., Li H., Hong G., Wu B., Qiu Q, & Lu Z. (2017). Crosstalk between Mitochondrial Fission and Oxidative Stress in Paraquat-Induced Apoptosis in Mouse Alveolar Type II Cells. International Journal of Biological Sciences, 13(7), 888-900.

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MIN6 Cell Viability Analysis

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MIN6 cells were seeded into a 96-well plate (3000 cells/well) and serum starved in RPMI1640 medium for 24 h. The MIN6 cells were then exposed to 50 ng/mL ∆nFGF1 or FGF1^WT for 24 h or exposed to 33 mM HG and 0.5 mM PA in the presence or absence of 50 ng/mL ∆nFGF1 for 24 h. Cell Counting Kit-8 (CCK-8, Jiancheng Bioengineering Institute, China) was used to assess cell viability according to the manufacturer's instructions. In brief, cells treated as described above were incubated with 10 μL of CCK-8 solution at 37°C for 1 h. The optical density (OD) value was detected by a microplate reader (Thermo, USA).

Chen Q., Chen X., Jia Z., Du Y., Zhang S., Xu W., Pan B., Lou J., Zhou J., Zhou J, & Sun J. (2022). ∆nFGF1 Protects β-Cells against High Glucose-Induced Apoptosis via the AMPK/SIRT1/PGC-1α Axis. Oxidative Medicine and Cellular Longevity, 2022, 1231970.

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Cell Viability Assay with CCK-8

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Cell viability was determined by the Cell Counting Kit-8 (CCK-8) assay (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China). The experiment was divided into normal cell and LAB treatment groups, and the multiplicity of infection (MOI) was set to three gradients, 1, 2, and 3, with each well performed in sextuplicate. IPEC-J2 cells (1 × 10⁵ cells/well) were seeded into 96-well tissue culture plates with 100 μL per well. After cells grown in a humidified chamber at 37 °C under 5% CO₂, collected that it reached 80% confluence, CCK-8 was used to stain the cells for about 2, 4, 12, and 24 h after being treated by ZA3, respectively. The optical density was measured at 450 nm using a microplate spectrophotometer.

As: experimental well (including cell-containing medium, CCK-8 and ZA3);
Ac: control well (including cell-containing medium and CCK-8);
Ab: blank well (cell culture medium without cells, CCK-8).

Wang W., Ma H., Yu H., Qin G., Tan Z., Wang Y, & Pang H. (2020). Screening of Lactobacillus plantarum subsp. plantarum with Potential Probiotic Activities for Inhibiting ETEC K88 in Weaned Piglets. Molecules, 25(19), 4481.

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Evaluating Al2O3 NPs Cytotoxicity

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Cellular viability was evaluated by a CCK-8 proliferation assay using a Cell Counting Kit-8 (Nanjing Jiancheng Bioengineering Institute, China). HBE cells were plated at a density of 1 × 10⁴ per well in a 96-well plate and treated with 0, 12.5, 25, 50, 100, 250, 500, 1 000 μg/ml (corresponding to 0, 3.91, 7.81, 15.62, 31.25, 78.13, 156.25, 312.5 μg/cm²) Al₂O₃ NPs with eight biological replicates for each concentration. 10 μL of CCK-8 was added to each well, cells were incubated for 4 h at 37 °C, and the absorbance was determined at 450 nm. Cell viability affected by Al₂O₃ NPs were monitored every 24 h for up to 7 days.

Li X., Zhang C., Zhang X., Wang S., Meng Q., Wu S., Yang H., Xia Y, & Chen R. (2016). An acetyl-L-carnitine switch on mitochondrial dysfunction and rescue in the metabolomics study on aluminum oxide nanoparticles. Particle and Fibre Toxicology, 13, 4.

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CCK-8 Assay for Cell Viability

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The Cell Counting Kit‐8 (CCK‐8) reagent (Nanjing Jiancheng Bioengineering) assay has the advantages of excellent repeatability and low cytotoxicity. HaCaT cells at the logarithmic (log) phase of 4–5 generations were harvested and inoculated into 96‐well plates using 100 μL of (4–5) × 10⁴ cells/mL.
¹⁴ After the cells were cultured at 37°C for an overnight period in 5% CO₂, the 96‐well plates were incubated with varying concentrations of LMWP. After 24 h of interaction between LMWP and the cells, CCK‐8 reagent was added at a concentration of 10% (V/V), and the cells were incubated in the cell incubator (bluepard) for approximately 2 h. Finally, the OD value at 450 nm was determined using a microplate reader (MultiSKAN), and each concentration was tested in five replicates. The cell proliferation rate is calculated strictly based on the following formula
¹⁵ (link):

Cell viability % = \frac{Experimental Group O D - Blank Group O D}{Control group O D - Blank Group O D} \times 100 %

Chen Z., Cao P., Zhu T., Yan C., Zheng Z, & Yao H. (2024). Plum blossom low molecular weight polypeptide protects HaCaT cells against UVB‐induced oxidative damage in vitro and underlying mechanism. Skin Research and Technology, 30(2), e13582.

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Neurotoxicity of Lead and Selenium/Lead

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The IC50 of chicken embryonic neurocytes in the Pb and Se/Pb groups for 48 hours was measured using cell counting kit-8 produced by Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer's instructions. In the Pb group, Pb concentration was 2, 4, 6, 8, 10, 12, 14, 16, and 18 × 10⁻⁷ mol/L, respectively. In the Se/Pb group, Pb concentration was 2, 4, 6, 8, 10, 12, 14, 16, and 18 × 10⁻⁷ mol/L, respectively; and Se concentration was 10 × 10⁻⁸ mol/L.

Zhu Y., Jiao X., An Y., Li S, & Teng X. (2017). Selenium against lead-induced apoptosis in chicken nervous tissues via mitochondrial pathway. Oncotarget, 8(64), 108130-108145.

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Assessing PC12 Cell Viability

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Cell viability was determined by Cell Counting Kit-8 (CCK-8)^{34 (link)} (Jiancheng, Nanjing, China). PC12 cells were placed in 96-well plates at a density of 1 × 10⁴ cells per well and in a volume of l80 μL. The plates were cultured for 24 h, and 20 μL of samples were added to the experimental groups. The control and model groups were added 20 μL of culture medium. After 24 h of incubation, the culture medium was replaced with 100 μL medium containing 400 µM CoCl₂ while the control group was replaced with 100 μL new medium^{21 (link)}. After 12 h treatment, 10 µL CCK-8 solution was added to each well and the plates were incubated at 37 °C for an additional 2 h. The absorbance was measured at 450 nm using Biotek microplate reader (Winooski, VT, USA) and the background absorbance was excluded by performing blank corrections. Cell viability was expressed as a percentage of the untreated group (control = 100%).

Zhou F., Zhao Y., Li M., Xu T., Zhang L., Lu B., Wu X, & Ge Z. (2017). Degradation of phenylethanoid glycosides in Osmanthus fragrans Lour. flowers and its effect on anti-hypoxia activity. Scientific Reports, 7, 10068.

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Cell Proliferation Assay and Markers

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The proliferation of NRK-49F cells was detected by Cell Counting Kit-8 (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's protocol. In addition, the expression of the proliferation-associated protein PCNA and fibroblast-specific protein Fsp-1 was detected by western blot or immunofluorescence, which can reflect the proliferation of fibroblasts.

Zhao S., Li W., Yu W., Rao T., Li H., Ruan Y., Yuan R., Li C., Ning J., Li S., Chen W., Cheng F, & Zhou X. (2021). Exosomal miR-21 from tubular cells contributes to renal fibrosis by activating fibroblasts via targeting PTEN in obstructed kidneys. Theranostics, 11(18), 8660-8673.

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