The SW480 cells were seeded in six-well plates (3.5 × 105 cells/well) for 24 h and then treated with BBR for another 24 h. The cells were detached with 0.05% trypsin, washed three times with cold PBS, and centrifuged at 1500 rpm for 5 min at 4 °C. Subsequently, SW480 cells were gently suspended in the binding buffer containing 5 µL (10 µg/mL) Annexin V-FITC for 15 min in the dark, then incubated with 10 µL of PI (20 µg/mL) for another 5 min. The cells were immediately analysed by flow cytometry (BD FACSCanto II) (Tian et al. 2016).

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