Flow cytometry

To detect apoptotic cells, the pre-treated cells were harvested after trypsinization using EDTA-free trypsin, washed twice with PBS, and stained with the Annexin V-APC/7-AAD apoptosis kit (Multiscience, China) according to the manufacturer's instructions. The apoptotic cells were examined by flow cytometry (BD Bioscience, USA), and the data was analyzed with FlowJo X 10.0.7 software. For cell cycle analysis, the cells were harvested at about 80% confluency and fixed with cold 70% ethanol at -20°C overnight. After discarding the ethanol, the cells were washed with PBS, and incubated with DNA staining solution (Multiscience, China) for 30 minutes at room temperature. The DNA content were measured by flow cytometry, and the data was analyzed using ModFit software.

H460/CDDP cells were logarithmically cultivated and seeded into 6-cm dishes at a density of 2 × 10⁴ cells/ml. After treatment with the combination of d-borneol (0.5, 1, 2, and 4 μg/ml) and CDDP (10 μg/ml) for 24 h, H460/CDDP cells were collected, washed with PBS, and fixed with ethanol for 24 h. Then the cells were stained with propidium iodide (PI) for 30 min and protected from light. Flow cytometry was utilized to detect changes in cell cycle distribution (BD Biosciences, CA, USA) and analyzed by FlowJo V10 software [28 (link)].

Reactive Oxygen Species Detection Kit was exploited to measure the ROS formation in the retinoblastoma cells of HXO-Rb44 incubated with the Na(I) complex on the basis of protocol [26 (link)]. The retinoblastoma cells of HXO-Rb44 were harvested and planted in 6-well plates (2 × 10⁵ cells/well) and then seeded at 5% CO₂ and 37°C. The wells were added with Na(I) complex (1×IC₅₀) and then the wells were incubated for one day. ROSup and PBS were respectively employed as positive and negative control. After that, discarding the medium and cleaning the cells 3 times via the PBS, then the cells were incubated by utilizing H2DCF-DA dye (20 μM) for twenty minutes in darkness. The flow cytometry (BD Via, New Jersey, USA) and FlowJo7.6 were exploited to measure and analyze the absorbance of samples at 488 and 530 nm excitation wavelength. Each research was implemented in triplicate.

flow cytometry assay was carried out for cell apoptosis assessment. In brief, different treated A549 and H1975 cells were harvested and subjected to apoptosis test using Annexin V (FITC)/propidium iodide (PI) Apoptosis Detection Kits (Dojindo, Japan). Cell apoptosis rate was detected by using flow cytometry (Beckman Coulter, CA, U.S.A.) and analyzed using FlowJo 7.6 software (FlowJo LLC, Ashland, OR, U.S.A.).