Apoptosis Measurement in SKOV3-TR Cells

To measure apoptosis, SKOV3-TR cells (3 × 10³) were seeded on black 96-well plates 24 hrs prior to the experiment. Liposomes were sterile filtered through 0.2-µm filters and incubated with the cells for 24 hrs and washed off. After a further 24 hrs, three fluorescent dyes: Hoechst 33342 (5 µg/mL), Yo-Pro (0.063 µg/mL) and propidium iodide (PI) (1 µg/mL) were added to stain live cell nuclei, cells in early apoptosis (slight membrane permeability), and late apoptosis/necrosis, respectively. After incubation at 37°C for 30 minutes, the cells were analyzed in situ using the iCyte^® laser scanning cytometer (CompuCyte Corp. Westwood, MA)(39 (link), 40 (link)). Excitation/emission wavelengths used were 405/440 nm with a 30-nm bandwidth for Hoechst, 488/515 nm with a 30-nm bandwidth for Yo-Pro and 488/635 nm for propidium iodide. All data analyses were carried out using the iCyte software (Version 3.4). Alternatively, DNA content distributions were measured by propidium iodide staining. Upon treatment with liposomes (50 nM, PCT; 40 nM, XR) for 18 hrs, the cells were permeabilized by 70% ethanol overnight, stained with PI/RNase buffer (BD Biosciences), and detected by an LSR II Fortessa cell analyzer. The cell cycle distributions were further processed by FlowJo software (38 (link)).