2.5. Annexin V/PI Assay and Analysis of Cell Apoptosis

Cancer cells were cultured in 6-well plates with PAA (0.2 mM) for 24 h or DMSO. The cells were trypsinized to be single cells and washed with 1× PBS. The single cells (1 × 10⁵) were counted and suspended with 100 µL of 1× Annexin V binding buffer. We added 5 µL of FITC Annexin V solution and 5 µL of propidium iodide staining solution to each samples. Then, those were incubated for 15 min at room temperature. After washing with 1× Annexin V binding buffer, the cell pellet was analyzed using an Accuri C6 flow cytometer (BD, San Jose, CA, USA). Nuclear staining of MDA-MB-231 cells was performed by Hoechst 33342 dye. Cancer cells were treated with PAA (0.1 mM) for 24 h, and incubated with Hoechst 33342 dye (10 mg/mL) solution for 30 min at 37 °C. The dye-stained cells were observed using a fluorescence microscope (Lionheart FX, Biotek, Winooski, VT, USA).