Lsrfortessa 6 laser

Five milliliters of a solution of human HepG2 hepatoma cells with the concentration of 5 × 10⁵ /mL was added in the tissue culture flask and incubated for 12 h. When cells were attached on the bottom of flask, 5 mL of 100 μg/mL solution of the samples (D-NP or N-NP) or control (no nanoparticles) was added after removal of the media and incubated for 5 h. The cells in each flask were then trypsinized and washed with PBS buffer by centrifugation and redispersion in 1 mL of fresh PBS buffer in the microtubes. Ten micromolar DCF-DA solution was added into each microtube and incubated at 37 °C for 30 min. Then, the cells were irradiated with X-ray for 4 Gy by placing the microtubes in the center of the X-ray irradiator for the radiation. The cells were incubated at 37 °C for 15 min and analyzed with flow cytometry (LSRFortessa 6-Laser, BD Biosciences) at excitation 485 nm.

Five milliliters of a solution of human HepG2 hepatoma cells with the concentration of5 × 10⁵ /mL was plated on tissue culture flask and incubated for 12 h. When cells were attached on the bottom of flask, 5 mL of 100 μg/mL samples (D-NP or N-NP) was added after removal of the media and incubated for 5 h. For the treatment of X-ray radiation, the flask was placed onto the center of the shelf in an X-ray irradiator for the radiation treatment. The cells were collected after culturing further for 3 days. The cells were trypsinization and washed for twice by centrifugation at 1500 rpm and redispersion in fresh PBS to remove trypsin. After recentrifugation to wash cells and make cell pellets, cells were resuspended in the 100 μL of 1× Annexin-binding buffer. Five microliters of FITC Annexin V and 1 μL of 100 μg/mL propidium iodide solution were added to the cells and cell incubation was performed at room temperature for 15 min. Lastly, 400 μL of 1× Annexin-binding buffer was added to the cells and the stained cells were kept on ice before measurement with flow cytometry (LSRFortessa 6-Laser, BD Biosciences). The fluorescence emission was set at 530 nm (FL1) and >575 nm (FL3) and 50 000 events were counted to observe the population of cells in three groups: live cells, apoptotic cells, and dead cells.

Five milliliters of a solution of human HepG2 hepatoma cells with the concentration of5 × 10⁵ /mL was plated on tissue culture flask and incubated for 12 h. When cells were attached on the bottom of flask, 5 mL of 100 μg/mL of samples (D-NP or N-NP) were added after removal of the media and incubated for 24 h. The cells were then trypsinized and washed for twice by centrifugation at 1500 rpm and redispersed in the fresh PBS to remove trypsin. After recentrifugation for cell sedimentation, cells were fixed with ice-cold 80% ethanol aqueous solution and stored in −20 °C freezer for 12 h. After fixation, the fresh PBS was added to the cells and the cells were washed by centrifugation and redispersion in the PBS. In the resuspended cell pellet, PI staining solution was added and the cells were incubated for 20 min at 37 °C and covered with foil and stored at 4 °C before analysis with flow cytometer flow cytometry (LSRFortessa 6-Laser, BD Biosciences). The fluorescence emission was set at 488 nm (FL2) and 40,000 events were counted to observe the population of cells. The cells cycle of the PI stained cells was analyzed with FlowJo10.4.1: the phase fractions of G0/G1, S, and G2/M were determined by the curve fittings of the FlowJo software.