*4.8. Annexin V*/*PI-Based Flow Cytometric Analysis*

Annexin V assays were performed according to the manufacturer's protocol (BD Pharmingen, San Diego, CA, USA). Briefly, 10,000 cells were plated into 60 mm plates and treated with KJ-28d (10 µM) for 48 h. Cells were harvested and incubated with 4 µL allophycocyanin (APC)-conjugated annexin V (20 µg/mL) and 4 µL PI (50 µg/mL) for 15 min. Fluorescence analyses were performed using flow cytometry (CyFlow cube 6). Cells were classified as early apoptotic (annexin V-positive/PI-negative), late apoptotic/necrotic (annexin V-positive/PI-positive), necrotic/dead (annexin V-negative/PI-positive), and live (annexin V-negative/PI-negative). Flow cytometry data were analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA).
