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CCK-8 cell viability assay

HC Hui Che
LW Liping Wang
YR Yongxin Ren

Last updated date: Dec 23, 2021 Views: 1330 Forks: 0

original An abbreviated version of this protocol was published in eLife in Mar, 2020
p16 deficiency attenuates intervertebral disc degeneration by adjusting oxidative stress and nucleus pulposus cell cycle
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  1. In each group, a density of 5000/well NP cells was seeded in a 200μL growth medium in 96-well plates. （We recommend inoculating cells in wells near the center of the plate, as the medium in the outermost ring of wells tends to evaporate）
  2. After treatments for the indicated time in the paper, the medium was replaced with 90μl fresh medium mixing 10μl CCK-8 assay solution in each well. (Note: Do not introduce air bubbles to avoid interfering with OD value detection)
  3. Meanwhile, the only 90μl fresh medium mixed 10μl CCK-8 assay solution without cells was set as the blank control group.
  4. Incubate cells at 37°C in the dark for 3 hours. (Note: The optimal reaction time for CCK-8 is based on the specific degree of color development of the cells)
  5. The absorbance of each well was measured at 450 nm by an enzyme marker with gentle mixing on a shaker before reading.
  6. We calculated the cell proliferation using the absorbance of cells, which is As-Ab. (As = absorbance of experimental wells with cells, Ab = absorbance of blank wells without cells)
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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Che, H, Wang, L and Ren, Y(2021). CCK-8 cell viability assay. Bio-protocol Preprint. bio-protocol.org/prep1481.
  2. Che, H., Li, J., Li, Y., Ma, C., Liu, H., Qin, J., Dong, J., Zhang, Z., Xian, C. J., Miao, D., Wang, L. and Ren, Y.(2020). p16 deficiency attenuates intervertebral disc degeneration by adjusting oxidative stress and nucleus pulposus cell cycle. eLife. DOI: 10.7554/eLife.52570

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