Flow cytometer analysis

Cell apoptosis and decrease of mitochondrial transmembrane potential induced by melatonin or 5-Fu was determined by AnnexinV/PI (KeyGEN, Nanjing, China) and rhodamine (Beyotime, Shanghai, China) staining, respectively, followed by flow cytometer analysis (Beckman Coulter, California, USA) according to manufacturer's instructions. Also, caspase activity was measured by Caspase 3/7 Glo assay and Caspase 8 Glo assay (Promega, Madison, WI, USA) according to the manufacturer's protocol. The intracellular level of ROS was detected according to a previous report.^{42 (link)}

Cell apoptosis was determined by AnnexinV/PI (KeyGEN, Nanjing, China), followed by flow cytometer analysis (Beckman Coulter, California, USA) according to manufacturer’s instructions.
Caspase activity was measured by Caspase-Glo 3/7 Assay kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol.

Annexin V/PI assay was used to determine the apoptotic and necrotic cells. The A549 and MCF-7 cells were seeded on 6-well microplates at a density of 10⁶ cells/mL respectively. After 24 h incubation, the cells were treated with following concentrations of 0, 5, and 10 μM for compound 1. After 24 h, they were washed and re-suspended in PBS solution (500 μL). Then, Annexin V-FITC (5 μL) and PI staining solution (5 μL) were introduced to the mixture, and the incubation process was followed under the dark condition for 5 min at 25 °C. Finally, flow cytometer analysis (Beckman Coulter^®, Miami, FL, USA) was performed using an AnnexinV-FITC Apoptosis Detection Kit (Strong Biotech Corporation, Taipei, Taiwan) and Flowjo version 7.6.1. Software.

Propidium iodide (PI) staining was used to analyze DNA contents and cell cycle. Briefly, total CD4⁺ T cells were isolated from the MLN of naïve 6- to 8-week-old C57BL/6 mice using EasySep™ Mouse CD4⁺ T Cell Isolation Kit. Cells were cultured in 96-well plate at 2×10⁵ cells/well and stimulated with anti-CD3 (1 μg/ml) plus anti-CD28 (1 μg/ml) in the absence (DMSO control) or presence of 40 mg/L DH for 24 h or 48 h. Cells neither stimulated nor treated with DH were used as control (0 h). Alternatively, total CD4⁺ T cells were isolated from the CLN of IRBP-immunized mice (10 days after immunization) and stimulated with IRBP-loaded BMDCs in the absence (DMSO control) or presence of DH (10, 20, and 40 mg/L) for 48 h. After surface staining with FITC-anti-CD4 antibody, cells were fixed and incubated with staining buffer containing 50 µg/ml PI and 0.25 mg/ml RNase A (Beyotime) at room temperature for 30 min. Cell cycle distribution was determined using flow cytometer analysis (Beckman Coulter). The percentages of cells in the G0/G1, S, and G2/M phases were determined using Beckman software.

Cell apoptosis were determined by Annexin-V/PI (KeyGEN, Nanjing, China) staining, followed by flow cytometer analysis (Beckman Coulter, California, USA) according to the manufacturer's instructions. The experiment was triplicately repeated.

Cell apoptosis and cell cycle was evaluated by flow cytometry. Transfected cells were resuspended in binding buffer after wash with cold PBS. Suspended cells were incubated with Annexin V-FITC and propidium (PI) for 10–15 min at room temperature followed by flow cytometer analysis (Beckman, USA): AnnexinV-FITC (-) and PI (-), living cells; AnnexinV-FITC (+) and PI (-), early apoptotic cells; AnnexinV-FITC (+) and PI (+), late apoptotic cells and necrotic cells. Cells that were AnnexinV-FITC (+) /PI (-) or AnnexinV-FITC (+) /PI (+) cells were considered apoptotic cells. For cell cycle analysis, cells were fixed in 70 % ethanol at 4 °C overnight and stained with 5 µL PI for 45 min in the dark at room temperature. Then the stained cells were immediately analyzed by flow cytometry (Beckman, USA).