Flow cytometry analysis

The Annexin V‐FITC Apoptosis Detection Kit microscopy (C1062, Beyotime Biotechnology) was applied under the manufacturer's instructions for apoptosis detection. In brief, primary cardiomyocytes were moulded, harvested after trypsinization treatments and washed twice with PBS; the cells were resuspended in 195 µL binding buffer, stained with Annexin V‐FITC and then subsequently incubated with both 5 µL of Annexin V‐FITC and 10 µL of the PI working substrate for 20 minutes at room temperature in the dark. Cellular fluorescence was measured by flow cytometry analysis (Becton‐Dickinson, USA). In the early stages of apoptosis, cells were Annexin V‐positive, while cells that were both PI‐ and Annexin V‐positive were at the later stages, whereas cells that were only PI‐positive were necrotic.

For cell cycle analysis, the cells were collected 48 h after electrical therapy. After washing with phosphate-buffered saline solution, 1 mL of precooled 70% ethanol was added, and the mixture was incubated overnight at 4 °C. Then, the cells were collected again by low-speed centrifugation and suspended in 500 μL of flow cytometry (FACS) buffer supplemented with 2.5 μL of 1.0 mg/mL RNase at room temperature for 15 min. Then, the cells were incubated with 25 μL of propidium iodide (1 mg/mL) at room temperature in the dark for 15 min. The distribution of the cell cycle was detected by flow cytometry analysis (Becton Dickinson, Franklin Lakes, NJ, USA).
In the apoptosis assay, the cells were placed into 6-well plates at a density of 5 × 10⁵/well and grown overnight at 37 °C with 5% CO₂. The electrical stimulation of SFB-TENGs with different outputs was applied after rectification for 1 h/day in each treatment group. After 48 h, the cells were collected for apoptosis assessment with Annexin-V (FITC) and PI kits (KeyGEN BioTECH, China) and subsequently analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA). Furthermore, the treated cells were stained with calcein and PI (KeyGEN BioTECH, China) after 48 h. Then, a fluorescence microscope was used to obtain optical images.

The protection of terpenylated wogonin derivatives on LPS-induced reactive oxygen intermediates productions was measured by DCFH (2′,7′-dichlorofluorescin) oxidized fluorescent products retained in RAW264.7 cells. Cells incubated with 50 μM terpenylated wogonin derivatives and 100 ng/mL LPS were loaded with DCFH-DA (dichlorofluorescin diacetate) dye for detecting the amount of intracellular reactive oxygen species. Analysis were performed at 3, 6, 24, and 48 h. Upon a 10 min incubation with DCFH-DA dye loading at dark, room temperature, cells were quickly immersed into ice-cold water incubation prior to flow cytometry analysis (Becton Dickson, Franklin Lakes, NJ, USA). Data analysis was completed by the CellQuest software (Becton Dickson, Franklin Lakes, NJ, USA). The percentage of cells population exerts fluorescence more than the second log values were considered and the geometrical means of fluorescent intensity were recorded.