For CCK-8 assay, CRC cells were seeded into 96-well plates at a density of 1 × 103 per well. Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) was added into each well at indicated time points. After incubation at 37 °C for 2 h, the absorbance was measured using an automatic microplate at 450 nm.
For EdU (5-ethynyl-2′-deoxyuridine) assay, exponentially growing cells plated on 6-well plates (Corning) were labelled as described [21 (link)]. Then EdU-labeled or unlabeled LoVo and HCT116 cells were cultured into a 96-well plate at 2 × 103 cells/well in McCoy’s 5A medium added with 10% FBS at 37 °C. At 0, 24, 48, 72, and 96 h, 10 μl of Cell Titer-96 reagent (Promega Inc., Madison, WI) was supplemented to each well. After 1 h of further incubation at 37 °C, the cells were scanned at the wavelength of 490 nm in a plate reader (Molecular Devices Corp., Sunnyvale, CA).
For EdU (5-ethynyl-2′-deoxyuridine) assay, exponentially growing cells plated on 6-well plates (Corning) were labelled as described [21 (link)]. Then EdU-labeled or unlabeled LoVo and HCT116 cells were cultured into a 96-well plate at 2 × 103 cells/well in McCoy’s 5A medium added with 10% FBS at 37 °C. At 0, 24, 48, 72, and 96 h, 10 μl of Cell Titer-96 reagent (Promega Inc., Madison, WI) was supplemented to each well. After 1 h of further incubation at 37 °C, the cells were scanned at the wavelength of 490 nm in a plate reader (Molecular Devices Corp., Sunnyvale, CA).
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