4.8. Apoptosis Analysis and Cell Cycle Analysis by Flow Cytometry

Annexin V/propidium iodide (PI) dual-staining method is a sensitive assay for quantitative determination of apoptotic cells [35]. SKBR3 cells were seeded in 6-well plate at a density of 2 × 10⁴ cells/well, and incubated for 24 h for adhesion. Afterwards, old culture media was replaced with fresh culture media, and cells were treated with curcumin or Cur-SLNs for 24 h. Later, the cells were washed twice by PBS, and stained with Annexin V and PI at 37 °C for 30 min. The cells were then analyzed by flow cytometry (BD Bioscience, San Jose, CA, USA) for measuring the proportion of apoptotic cells.

Propidium iodide (PI) staining of cells and subsequent analysis by flow cytometry was carried out to analyze the cell cycles. After overnight plating in 6-well per well, cells were incubated with 20 μM curcumin, Cur-SLNs for 24 h. The cells were harvested with trypsin 24 h after treatment, washed twice with PBS, and 1.0 × 10⁶ cells were suspended in the binding buffer of the Annexin V kit. Then, cells were resuspended in precooled 70% ethanol, and fixed by storage on ice for at least 2 h. After washing with ice-cold PBS, to prevent inadvertent staining of dsRNA, the cells were treated with 50 μL RNaseA at 37 °C for 30 min. Ultimately, the cells were washed with PBS and stained with propidium iodide for 30 min at room temperature. Next, the flow cytometry analysis was carried out for 10,000 events per sample through FL2-A band-pass filter (PI) using flow cytometry.