Evaluating Endothelial Cell Apoptosis

The influence of EBC samples on murine endothelial C-166 cell line was also assessed using FITC (fluorescein isothiocyanate)-labelled Annexin V/propidium iodine (PI) assay. The test was performed according to a modified manufacturer’s protocol (FITC Annexin V Apoptosis Detection Kit, BD Pharmingen, Franklin Lakes, NJ) [11 (link)]. The cells were incubated with medium alone (baseline control), EBC samples or 0.02% Triton X-100 (negative control) for 1 or 4 h. Then, they were subsequently washed twice with cold PBS and incubated with FITC-labelled Annexin V and PI for 15 min. The test was repeated three times. The presence of Annexin V- and/or PI-positive cells was assessed using Nikon Eclipse E800 fluorescent microscope with Nikon Coolpix 995 digital camera (Nikon, Japan).