To study the radiosensitization effect of HA-Bi2O3 NPs, SMMC-7721 cells were seeded in six-well plates at a density of 4.0×104 cells/well and allowed to grow for 12 h and divided into six groups (control, HA-Bi2O3 NPs, radiation, radiation +50 μg/mL HA-Bi2O3 NPs, radiation +100 μg/mL HA-Bi2O3 NPs, and radiation +200 μg/mL HA-Bi2O3 NPs). When cells had grown to 80% in plates, the first group received no treatment, the second one was incubated with 200 μg/mL HA-Bi2O3 NPs for 24 h, the third one was just irradiated at 6 Gy, and the fourth to sixth ones were irradiated at 6 Gy, and at the same time, incubated with different concentrations of HA-Bi2O3 NPs (50, 100, and 200 μg/mL) for 24 h, respectively. After that, FDA and PI working buffer was added for cell staining. The fluorescence of stained cells was observed under a fluorescence microscope; live cells showed green color, and dead ones exhibited red color.
Furthermore, cells treated by different treatments were washed three times with PBS, digested, collected, and centrifuged at a speed of 2,000 rpm for 5 min, and then fixed with 70% ethanol at −20°C overnight followed by PI staining. DNA fragmentation was quantified by the fluorescence intensity of PI on a Beckman Coulter Epics XL MCL flow cytometer (BD Accuri C6) and analyzed by software (Flowjo 7.6.2) to clearly understand the cell cycle distribution and apoptosis.
Furthermore, cells treated by different treatments were washed three times with PBS, digested, collected, and centrifuged at a speed of 2,000 rpm for 5 min, and then fixed with 70% ethanol at −20°C overnight followed by PI staining. DNA fragmentation was quantified by the fluorescence intensity of PI on a Beckman Coulter Epics XL MCL flow cytometer (BD Accuri C6) and analyzed by software (Flowjo 7.6.2) to clearly understand the cell cycle distribution and apoptosis.