Jc 1 mitochondrial membrane potential dye

Here, L02 and HepG2 cells were plated at a density of 2 × 10⁵ cells/well in 96-well plates. Then, they were treated with bavachin (17, 34, and 68 μmol/L), psoralidin (11.5 23, and 46 μmol/L), bavachinin (15, 30, and 60 μmol/L), neobavaisoflavone (23.5, 47, and 94 μmol/L), and bakuchiol (13, 26, and 52 μmol/L) for 24 h. Then Hoechst 33342, eBioscience™ JC-1 Mitochondrial Membrane Potential Dye, CellROX Deep Red, and HCS LipidTOX™ Green Phospholipidosis Detection Reagent kits were used to characterize their cell counts, nuclear area, MMP, ROS, and intracellular lipid. Consequently, multi-parameter cytotoxicity was analyzed through high-content screening (HCS) analysis (IN Cell Analyzer 2500 HS; Cytiva, Marlborough, MA, USA). Then, a 20× objective was used to collect all images. Three independent wells were examined for each treatment, and 16 fields per well were captured during the analysis. Lastly, HCS analysis was performed using Automated Image and Cell Analysis software (IN Cell Analyzer 2000; USA).

After 24 h PCC treatment of both cancer and normal cells, 10 µg/ml JC-1 mitochondrial membrane potential dye (eBioscience, San Diego, CA) were added to the live cells followed by incubation for 30 min at 37 °C. Cells were then fixed in 4% paraformaldehyde, permeabilized by 0.25% Triton X-100, quenched with 50 mM ammonium chloride and blocked with 5% BSA in PBS overnight, followed by probing with 5 µg/mL anti-cytochrome c antibody (Alexa Fluor 488; Abcam Inc., Cambridge, MA, USA) for 1 h. 10 µg/mL DY350-Phalloidin and 4 µg/mL Hoechst 33342 (Thermo Scientific, Hudson, NH, USA) were added into the staining solution and cells were incubated at room temperature for 20 min. Finally, cells were washed with PBS and coverslips were mounted using polyvinyl alcohol mounting medium (Fluka Analytical, Milan, Italy). Fluorescence was analysed using the Radiance 2100 confocal microscope (Bio-Rad, Hercules, CA, USA). Noise reduction was attained by Kalman filtering during application and data were analyzed with Data Viewer version 3.0.

After 24 h PCC treatment of both cancer and normal cells, 10 μg/ml JC-1 mitochondrial membrane potential dye (eBioscience, San Diego, CA) were added to the live cells followed by incubation for 30 min at 37 °C. Cells were then fixed in 4% paraformaldehyde, permeabilized by 0.25% Triton X-100, quenched with 50 mM ammonium chloride and blocked with 5% BSA in PBS overnight, followed by probing with 5 μg/ml anti-cytochrome c antibody (Alexa Fluor 488, Abcam Inc., Cambridge, MA, USA) for 1 h. 10 μg/ml DY350-Phalloidin and 4 μg/ml Hoechst 33342 (Thermo Scientific, Hudson, NH, USA) were added into the staining solution and cells were incubated at room temperature for 20 min. Finally, cells were washed with PBS and coverslips were mounted using polyvinyl alcohol mounting medium (Fluka Analytical, Milan, Italy). Fluorescence was analyzed using the Radiance 2100 confocal microscope (Bio-Rad, Hercules, CA, USA). Noise reduction was attained by Kalman filtering during application and data were analyzed with Data Viewer version 3.0.