Apoptosis detection

HeLa cells were cultured to a density of 2×10⁵ cells/well in 6-well plates and were subsequently treated with 0, 20, 40 and 80 µM 23,24-dihydrocucurbitacin B for 24 h. Cells were then stained with DAPI for 20 min at room temperature. The cells were then fixed with 70% methanol at −20°C overnight and observed using fluorescence microscopy (magnification, ×200). A similar procedure was followed for Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) (Sigma-Aldrich; Merck KGaA) staining; cells were stained with annexin V/PI and investigated using a flow cytometer, (BD Biosciences, San Jose, CA, USA) following the manufacturer's protocol and BD FACSuite software version 1.0 for analysis.