Cell counting kit 8 cck 8

Manufactured by Apexbio

Sourced in United States, China

The Cell Counting Kit-8 (CCK-8) is a colorimetric assay used to measure the number of viable cells in cell proliferation and cytotoxicity assays. It utilizes a water-soluble tetrazolium salt that is reduced by dehydrogenases in viable cells to produce a colored formazan dye, which can be quantified by absorbance measurement.

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CCK-8 (124 citations) CCK-8 kit (51 citations)

75 protocols using cell counting kit 8 cck 8

Cytotoxicity and Hepatic Effects of Sch B

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Sch B (CAS #61281-37-6) was purchased from MedChemExpress, with purity 99.86%, molecular weight 400.46, and molecular formula C₂₃H₂₈O₆. To examine the cytotoxicity of Sch B, 1×10⁴ cells were seeded in 96-well culture plates in culture medium for 24 h, then the culture medium was switched to medium containing 0, 1, 10, 25, 50, 100, 200 μM Sch B, and cells were cultured in a humidified atmosphere at 37°C with 5% CO₂ for 72 h. The effects of Sch B on cell viability were qualified by using the Cell Counting Kit-8 (CCK-8; ApexBio Technology LLC).
Cytotoxicity test results allowed for determining the appropriate concentration of Sch B. To investigate the optimal treatment concentration and duration of Sch B, different concentrations were added at different stages of the induction process. QPCR was used to assess the expression of hepatic marker genes to evaluate the effect of Sch B.

Jin M., Yi X., Zhu X., Hu W., Wang S., Chen Q., Yang W., Li Y., Li S., Peng Q., Pan M., Gao Y., Xu S., Zhang Y, & Zhou S. (2024). Schisandrin B promotes hepatic differentiation from human umbilical cord mesenchymal stem cells. iScience, 27(2), 108912.

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Measuring Cell Proliferation with CCK-8 and EdU

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Cell proliferation was detected using the Cell Counting Kit-8 (CCK-8, #K1018, APExBIO, USA) and the EdU cell proliferation assay kit (#C0071S, Beyotime Biotech, China), following the manufacturer’s instructions. The colony-formation assay was also performed to assess cell proliferation. 5000 cells were plated per well in triplicate in 6-well plates. The culture medium was changed every 3 days. Once visible clones were observed, each well was washed with PBS three times, fixed with methanol for 30 minutes at room temperature, and then stained with 0.05% crystal violet for 30 minutes. After washing, the colonies were counted and imaged.

Mao J., Tao Y., Wang K., Sun H., Zhang M., Jin L, & Pan Y. (2024). Identification of hub genes within the CCL18 signaling pathway in hepatocellular carcinoma through bioinformatics analysis. Frontiers in Oncology, 14, 1371990.

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Cell Viability and Proliferation Assay

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Cell viability was assessed with a Cell Counting Kit-8 (CCK-8) (APExBIO-K1018) and 5-ethynyl-2′-deoxyuridine (EdU) imaging kits (UE, China). A total of 2 × 10³ MODE-K cells per well were cultured in 96-well plates and treated as described above. The CCK-8 and EdU assays were performed according to the manufacturer's recommended procedures.

Chang Y., Zhang Y., Jiang Y., Zhao L., Lv C., Huang Q., Guan J, & Jin S. (2022). From Hair to Colon: Hair Follicle-Derived MSCs Alleviate Pyroptosis in DSS-Induced Ulcerative Colitis by Releasing Exosomes in a Paracrine Manner. Oxidative Medicine and Cellular Longevity, 2022, 9097530.

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Antibody-based protein detection assay

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Antibodies were used as follows: DAZAP1 (182558, Abcam, UK); FLAG (14793, Cell Signaling Technology, USA); GAPDH (60004-I-Ig, Proteintech, China); KITLG (sc-13126, Santa Cruz, USA); Puromycin was purchased from Merck KGaA (Darmstadt, Germany). Cell counting kit-8 (CCK8) was acquired from Apexbio, USA (K1018).

Zhou Y., Huangfu S., Li M., Tang C., Qian J., Guo M., Zhou Z., Yang Y, & Gu C. (2022). DAZAP1 facilitates the alternative splicing of KITLG to promote multiple myeloma cell proliferation via ERK signaling pathway. Aging (Albany NY), 14(19), 7972-7985.

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Cytotoxicity Assessment of SP2509 on Vero Cells

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The cytotoxic effects of SP2509 (GlpBio, USA) on Vero cells were assessed using the Cell Counting Kit-8 (CCK-8, APExBIO, USA). Briefly, 1 × 10⁴ Vero cells were seeded per well in 96-well plates containing different concentrations of SP2509. After 48 h, 10 μL of CCK-8 was added to each well, and the samples were incubated for 1~4 h. The absorbance of each well was measured at 450 nm using a microplate reader (BioTek, USA). All assays were performed in triplicate.

Zhao X., Zhang Y., Qu S., Tang W., He T., Li P, & Zheng X. (2024). SP2509, a specific antagonist of LSD1, exhibits antiviral properties against Porcine epidemic diarrhea virus. BMC Veterinary Research, 20, 187.

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Optimizing Muscle Cell Proliferation with Leucine

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Muscle cells viability were evaluated by Cell Counting Kit-8 (CCK-8, APExBio, USA) in order to analyze the effect of different concentrations of leucine on the proliferation of muscle cells to confirm the optimal leucine concentration, and to carry out follow-up experiments (Figure 1). The leucine (S20044, Yuanye, China) was designed as 7 concentration gradients of 0 (the media), 10, 20, 40, 80, 160, 320 mg/L. This leucine was soluble in the medium used in this experiment. The cell suspension (100 μL/well) was seeded in a 96-well plate, and after the cells adhered, the cell culture medium was replaced, and equal volumes of leucine with different concentrations were added for culture. After culturing for 24 h, 10 μL of CCK-8 solution was added to each well, placed in a 37°C cell incubator for 3 h, and then taken out, and the absorbance value of each well at 450 nm was detected with a microplate reader, with 4 replicates in each group.

Wang M.M., Guo H.X., Huang Y.Y., Liu W.B., Wang X., Xiao K., Xiong W., Hua H.K., Li X.F, & Jiang G.Z. (2022). Dietary Leucine Supplementation Improves Muscle Fiber Growth and Development by Activating AMPK/Sirt1 Pathway in Blunt Snout Bream (Megalobrama amblycephala). Aquaculture Nutrition, 2022, 7285851.

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MCL Radiosensitization in NSCLC

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Cells were treated with MCL (0~60 μM) for 6 h to assess the cell killing effect of MCL on NSCLC lines. Then the cell viability was measured after pretreatment with MCL (6 h before IR) pulse with IR to assess the radiosensitizing effect of MCL. For hypoxic exposure, four hours after MCL administering, the cells were moved into hypoxystation for additional 2 h before IR. The MCL-containing medium was replaced with normal culturing medium after IR and then cell viability was evaluated at 72 h after IR. Cell viability was measured with a Cell Counting Kit-8 (CCK-8, ApexBio, Houston, TX, USA). Two hundred microliters of CCK-8 solution was added to each well and incubated for 1 h at 37 °C. Absorbance at 450 nm was measured while using a Microplate Reader (Varioskan Flash, Thermo Fisher, Waltham, MA, USA). IC50 determination was performed using GraphPad Prism 7.0 software (GraphPad Prism Software, Inc., San Diego, CA, USA).

Kong P., Yu K.N., Yang M., Almahi W.A., Nie L., Chen G, & Han W. (2020). Micheliolide Enhances Radiosensitivities of p53-Deficient Non-Small-Cell Lung Cancer via Promoting HIF-1α Degradation. International Journal of Molecular Sciences, 21(9), 3392.

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Glycosaminoglycan ELISA and Cell Assays

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Vancomycin was purchased from Aladdin (Shanghai, China), Triton X100 and CH₃(CH₂)₁₁SO₄Na from Sinopharm Chemical Reagent Co., Ltd. (China), Pig glycosaminoglycan (GAG) ELISA Kit from Jianglai Biotechnology Co., Ltd (Shanghai China), Hyp assay kit from Beijing solarbio science & technology Co., Ltd. (China). Agar from Sangon Biotech Co., Ltd. (Shanghai, China), TRYPTONE from OXOID (Shanghai, China), and soya peptone from Sinopharm Chemical Reagent Co., Ltd. (China). DNA kit from CW Biotech (Beijing, China), Live/dead cell staining dye was purchased from BioVision (USA), and Cell Counting Kit-8 (CCK8) from APExBIO Technology LLC (USA).

Cai D., Chen S., Wu B., Chen J., Tao D., Li Z., Dong Q., Zou Y., Chen Y., Bi C., Zu D., Lu L, & Fang B. (2021). Construction of multifunctional porcine acellular dermal matrix hydrogel blended with vancomycin for hemorrhage control, antibacterial action, and tissue repair in infected trauma wounds. Materials Today Bio, 12, 100127.

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Cytotoxicity Evaluation of Hydrogels

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Cytotoxicity was determined using the Cell Counting Kit-8 (CCK8) (APExBIO Technology LLC, USA). The ADM and VADM hydrogels were immersed in DMEM (volume of hydrogel/volume of medium = 1:5) at 37 °C for 24 h. Collected the supernatant extract as the 100% leaching solution, then mixed with a certain proportion of DMEM medium to obtain 0%, 25% 50%, 75% leaching solution for further experiments. The NIH3T3 cells were seeded in a 96 well plate at a density of 3000 cells per well. After incubation at 37 °C for 24 h, the supernatant medium was removed from each well and 100 μL graded concentrations of leaching solution were added (0%, 25%, 50%, 75%, 100%, n = 5 for each concentration per day) for 1, 3, and 5 days. The NIH3T3 cells cultured with DMEM (0% leaching solution) was the control group. Subsequently, 10 μL of the CCK8 reagent was added to each well and quantified the cell metabolism after incubating for 1 h at 37 °C. The absorbance at an OD of 450 nm was measured using a spectrophotometer.

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LPS and Triton-X-100 induced cell responses

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Lipopolysaccharide (LPS) and Triton-X-100 were procured from Sigma-Aldrich (St. Louis, MO, USA). Cell Counting Kit-8 (CCK8) was secured from APExBIO Technology (Houston, USA). The commercial-specific complete Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12), penicillin, and streptomycin were acquired from GIBCO (Carlsbad, CA, USA). The ECL kit was provided by Tanon (Shanghai, China). In addition, 40, 6-diamidino-2-phenylindole (DAPI) was purchased from Beyotime (Shanghai, China). Primary and secondary antibodies used for western blotting were brought from Cell Signaling Technology (Danvers, MA, USA). Biotin-conjugated anti-Rabbit IgG antibody and DyLight 594-conjugated Avidin were picked up from Jackson ImmunoResearch (Pennsylvania, USA).

Chen S., Zhang Y., Niu X., Mohyuddin S.G., Wen J., Bao M., Yu T., Wu L., Hu C., Yong Y., Liu X., Abd El-Aty A.M, & Ju X. (2021). Coral-Derived Endophytic Fungal Product, Butyrolactone-I, Alleviates Lps Induced Intestinal Epithelial Cell Inflammatory Response Through TLR4/NF-κB and MAPK Signaling Pathways: An in vitro and in vivo Studies. Frontiers in Nutrition, 8, 748118.

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