2.5. Apoptosis Assay

The apoptotic activity was assessed using Annexin V-FITC/propidium iodide (PI) double-staining (APOAF-20TST, Merck, Darmstadt, Germany). The cells (1 × 10⁵) were seeded into a 12-well plate and treated with the PPN1 (10¹⁰ particles/mL) and PPN2 (10⁹ particles/mL) formulations. The NTC and Triton-X-100-treated cells were utilized as control groups. Following incubation of 24 h, the cells were harvested and stained, as per the manufacturer’s instructions. All samples were incubated for 15 min at 37 °C with 5 µL Annexin V-FITC and 5 µL PI diluted in 490 µL of binding buffers (50 mM HEPES, 700 mM CaCl₂, pH 7.4). Apoptotic and necrotic cells were immediately analyzed via flow cytometry (BD FACSVerse). The analysis was then performed on FACSuite^TM Version 8.0 software with cell populations selected using a forward scatter (FSC)/sideward scatter (SSC) dot plot, thereby excluding cell debris (Figure S1). The population gate was set for 50,000 events. These gated events were then further analyzed for Annexin V (FITC channel, laser 488 nm) and PI (PE channel, laser 617 nm). The percentage of cells in each quadrant was then recorded. The lower-left (LL) quadrant consisted of the viable population, while necrotic cells were observed in the upper-left (UL) and the upper-right (UR) quadrants. The apoptotic cells were distinguished in the lower-right (LR) quadrant.