Cell counting kit 8 (cck8)

Manufactured by Thermo Fisher Scientific

Sourced in United States, China

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

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63 protocols using cell counting kit 8 (cck8)

Cell Proliferation and Apoptosis Assay

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We used the Cell Counting Kit 8 (Gibco) assay to assess cell proliferation. We placed 2000 transfected cells per well into 96-well plates in triplicate wells for 5 days and detected cell proliferation at 1, 2, 3, 4, and 5 d after seeding according to the standard manufacturer’s instructions.
We used the Annexin V-APC/7-AAD Apoptosis Kit (MultiSciences; Hangzhou China) to detect apoptosis according to the manufacturer's instructions. In short, after washing in PBS, cells were incubated in 7-AAD staining solutions and APC Annexin-V in the dark at room temperature for 5 min. After incubation, the cells were collected by a C6 flow cytometer (BD, NY, USA) and FlowJo 10.4 software was used for analysis. Apoptotic cells population include 7-AAD- and APC Annexin-V + (undergoing apoptosis) cells and 7-AAD + and APC Annexin-V + (end of apoptosis or death) cells.

Liang Y., Ye F., Cheng Z., Ou Y., Zou L., Hu Y., Hu J, & Jiang H. (2021). Calculated identification of mutator-derived lncRNA signatures of genomic instability to predict the clinical outcome of muscle-invasive bladder cancer. Cancer Cell International, 21, 476.

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Cell Proliferation and Colony Formation Assays

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We employed Cell Counting Kit-8 assays (CCK-8; Gibco, Logan, Utah, USA) to quantify cellular proliferation. We seeded transfected cells into 96-well plates at a density of 5,000 cells/well in triplicate. We detected cell viability with the CCK-8 system at 0, 24, 48 and 72 h after seeding, following standard procedure.
For colony formation assays, we seeded transfected cells into 6-well plates at a density of 2000 cells/well and maintained them in DMEM containing 10% FBS for 10 days. We imaged colonies and counted them after fixing and staining them.

Jin M., Liu X., Wu Y., Lou Y., Li X, & Huang G. (2022). Circular RNA EPB41 expression predicts unfavorable prognoses in NSCLC by regulating miR-486-3p/eIF5A axis-mediated stemness. Cancer Cell International, 22, 219.

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Inflammatory Response of HUVECs to LPS Modulation

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HUVECs were placed in 96-well plates at a density of 5 × 10³ cells per well and then treated with lipopolysaccharides (LPS; 5, 10, 20, 40, or 80 μg/ml), GQP-EES (1, 2, 4, 8, 16, or 32 μg/ml), 5 μg/ml LPS in the presence or absence of GQP-EES (1, 2, or 5 μg/ml), aspirin (10 μg/ml) with 5 μg/ml LPS, or BoxA (10 μg/ml) with 5 μg/ml LPS or N4-Acetylcytidine (N4A) triphosphate sodium (1 mM) with 5 μg/ml LPS for 24 h. Cells were treated by the Cell Counting Kit-8 (CCK-8, Gibco) and measured their option density at 450nm using Synergy Microplate Reader (BioTek, Winooski, VT, USA).

Wei X., Zhang B., Wei F., Ding M., Luo Z., Han X, & Tan X. (2022). Gegen Qinlian pills alleviate carrageenan-induced thrombosis in mice model by regulating the HMGB1/NF-κB/NLRP3 signaling. Phytomedicine, 100, 154083.

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Dopamine Modulation Using Xanthan and Konjac

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Dopamine (DA) was bought from Engery Chemical (Shanghai, China). Xanthan gum (XG) and konjac glucomannan (KGM) were purchased from Aladdin (Shanghai, China). Dulbecco's modified Eagle medium (DMEM), phosphate buffered saline (PBS), fetal bovine serum (FBS), and cell counting kit-8 (CCK-8) were purchased from Gibco (Thermo Fisher Scientific). All reagents and materials were all analytical purity and were used as received without purification.

Zeng Q., Qian Y., Huang Y., Ding F., Qi X, & Shen J. (2021). Polydopamine nanoparticle-dotted food gum hydrogel with excellent antibacterial activity and rapid shape adaptability for accelerated bacteria-infected wound healing. Bioactive Materials, 6(9), 2647-2657.

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Cell Proliferation Evaluation Using CCK-8 Assay

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After treatment, cell proliferation was evaluated with the Cell Counting Kit-8 (CCK-8) assay (Gibco, Grand Island, NY, USA), as previously described [22 (link)]. The formazan crystals were dissolved in dimethyl sulfoxide (DMSO), and the absorbance was measured with a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at a wavelength of 450 nm.

Cai D., Hong S., Yang J, & San P. (2020). The Effects of microRNA-515-5p on the Toll-Like Receptor 4 (TLR4)/JNK Signaling Pathway and WNT1-Inducible-Signaling Pathway Protein 1 (WISP-1) Expression in Rheumatoid Arthritis Fibroblast-Like Synovial (RAFLS) Cells Following Treatment with Receptor Activator of Nuclear Factor-kappa-B Ligand (RANKL). Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 26, e920611-1-e920611-9.

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In Vitro and In Vivo Immunomodulation

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DOTAP and CHOL were purchased from Avanti Co., Ltd. (AL, USA; purity ≥ 98%). Dimethyl sulfoxide (DMSO) and lipopolysaccharide (LPS) were provided by Sigma-Aldrich (MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) with high glucose, DMEM-F12, penicillin-streptomycin, fetal bovine serum (FBS), MEM NEAA, HEPES, sodium pyruvate, trypsin, and phosphate-buffered saline (PBS) were provided by Thermo Fisher Scientific (Waltham, MA, USA). The APC-CD40, APC-CD80, APC-CD86, FITC-CD11c, PE-MHCII, and FITC-CD8 antibodies were purchased from BD Bioscience Co., Ltd. (USA). GM-CSF and IL-4 were obtained from Pepro Tech (USA). CD8 immunomagnetic bead kits were purchased from Intervention (USA). The Cell Counting Kit-8 from Gibco (USA) and Cyto Tox 96 Non-Radioactive Cytotoxicity Assay Kit was purchased from Promega (Japan).
Hepa1-6 cells were purchased from the Institute of Chinese Academy of Sciences and cultured in a humidified atmosphere of 5% CO₂ at 37°C. The cells were cultured in DMEM supplemented with 10% FBS and 100 μg/mL penicillin-streptomycin. C57BL/6 mice were purchased from Jinan Pengyue Laboratories. All animal studies were approved by the Ethics Committee of Liaocheng University. All animal procedures were performed in accordance with the guidelines of the Committee on Animal of Liaocheng University.

Zhang Y., Xie F., Yin Y., Zhang Q., Jin H., Wu Y., Pang L., Li J, & Gao J. (2021). Immunotherapy of Tumor RNA-Loaded Lipid Nanoparticles Against Hepatocellular Carcinoma. International Journal of Nanomedicine, 16, 1553-1564.

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Hypoxia Regulation of Angiogenesis

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1640 culture medium (Gibco), Opti-MEM (Gibco), FBS (Gibco), penicillin, streptomycin, trypsin, RIPA, and Cell Counting Kit-8 (Gibco), rabbit anti-rat HIF-1α (Novus Biologicals), mouse anti-rat VEGF (Novus Biologicals), mouse anti-rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Abcam), DNA Ladder (Solarbio, China), SonoVue (Bracco, Italy), HIF-1α shRNA were synthesized by the Genomics Institute, Sonitron 2000V (Nepa Gene, Japan), Microscope (Nikon, Japan), High Speed Freezing Microcentrifuge (SCILOGEX, Americ ), and Multiskan GO (Thermo Scientific, America).

Liao Y., Luo H., He Z., Kuang Y., Chen P., Zhang X., Chen J., Wen Q., Xie Y, & Ding S. (2019). A Combination of UTMD-Mediated HIF-1α shRNA Transfection and TAE in the Treatment of Hepatic Cancer. BioMed Research International, 2019, 1937460.

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Thapsigargin Cytotoxicity Assay in Cell Lines

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TG was diluted with DMEM to concentrations of 0, 0.5, 1, 2, 4, 8 and 16 µM. After incubation with TG for 48 h, the SW-13 and NCI-H295R cells were treated with 100 µL DMEM and 10 µL Cell Counting Kit-8 (Gibco, USA). The plates were incubated in the dark for 1 h at 37 °C. The optical density (OD) value was evaluated using a SynergyH1 reader (BioTek, USA) at 450 nm.

Wu L., Huang X., Kuang Y., Xing Z., Deng X, & Luo Z. (2019). Thapsigargin induces apoptosis in adrenocortical carcinoma by activating endoplasmic reticulum stress and the JNK signaling pathway: an in vitro and in vivo study. Drug Design, Development and Therapy, 13, 2787-2798.

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Cell Viability and Colony Formation Assay

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We used Cell Counting Kit-8 (CCK-8; Gibco) assay to assess cell viability. We seeded transfected cells into 96-well plates at a density of 2000 cells per well in triplicate wells. We detected cell viability at 0, 24, 48, and 72 h after seeding according to standard procedure.
For the colony formation assay, we seeded transfected cells into 6-well plates at a density of 2000 cells per well. Cells were maintained in DMEM medium containing 10% FBS for 10 days. We fixed and stained samples, and counted and imaged colonies.

Yang C., Li Q., Chen X., Zhang Z., Mou Z., Ye F., Jin S., Jun X., Tang F, & Jiang H. (2020). Circular RNA circRGNEF promotes bladder cancer progression via miR-548/KIF2C axis regulation. Aging (Albany NY), 12(8), 6865-6879.

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Cell Proliferation Assay with CCK-8

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We seeded 2000 T24 cells per well into 96-well culture plates for 5 days in triplicate wells. Cell Counting Kit 8 (Gibco) was used according to the manufacturer’s instructions. Then, 10 μL of CCK-8 reagent was added to each well and incubated for 1–2 h. The optical density (OD) values of each well were determined at 450 nm using a microplate reader.

Liang Y., Ye F., Xu C., Zou L., Hu Y., Hu J, & Jiang H. (2021). A novel survival model based on a Ferroptosis-related gene signature for predicting overall survival in bladder cancer. BMC Cancer, 21, 943.

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