Facsuite system

To determine the extent of apoptosis and necrosis, after treatments with F1, F2, and F3 (0.1 μg/ml) for 1, 5, and 24 h, respectively, 2 × 10⁵ cells were stained with fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide (Pi), using the Annexin V-FITC Apoptosis Detection Kit (Biolegend, San Diego, CA, United States #640914), following the manufacturer’s instructions. A total of 10,000 cells were analyzed, and apoptosis/necrosis was determined using the FACSVerse cytometer and FACSuite system (BD Biosciences). The data were analyzed using FlowJo software (v7.6.5). For cell cycle distribution, following the treatments, 2 × 10⁴ cells were fixed overnight at 4°C in 70% ethanol. The fixed cells were then washed with PBS and incubated with 1 ml solution containing 50 μg/ml Pi, and 0.5 µg RNase A, for 30 min, in the dark. Cell cycle distribution was assessed by flow cytometry using the FACSVerse cytometer and FACSuite system (BD Biosciences, Franklin Lakes, NJ, United States), and the data were analyzed using FlowJo software (v7.6.5).

NG97 cells were seeded in 24‐well plates (Corning, NY) at an initial density of 1.0 × 10⁵ cells per well and incubated at 37 °C for 72 h. The cells were then treated with F1, F2 or F3 (1 µg/mL), while the control cells were maintained in the medium. To determine the extent of apoptosis and necrosis, 1.0 × 10⁵ cells were stained with fluorescein isothiocyanate (FITC)–conjugated annexin V and propidium iodide (Pi), using the annexin V‐FITC apoptosis detection kit (#640,914; Biolegend, San Diego, CA) following the manufacturer’s instructions. A total of 10,000 cells were analyzed and cell apoptosis–necrosis was determined using the FACSVerse Cytometer and FACSuite system (BD Biosciences). The data were analyzed with FlowJo software (v7.6.5; Tree Star. Inc., Ashland, OR).