Annexin V Apoptosis Assay in HUVEC Cells

The HUVEC cells were seeded onto a 6-well plate (2.5 × 10⁵ cells per well) and cultured overnight. Next day, PDT was performed after 3 h of cell treatment with either sole 1 µM and 100 nM Zn-Pheide in serum-free culture medium or 1 µM Zn-Pheide in the presence of 250 µM HSA. Control cells were incubated in serum-free ECM medium, with or without 250 HSA, containing an appropriate concentration of DMSO. Heat-treated cells (55 °C for 20 min) served as a positive control. Thirty minutes after the PDT, the cells were harvested by trypsinization, washed with PBS, and suspended in Annexin V-binding buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) at a density of 10⁶ cells/mL. The cells were stained with Annexin V, Pacific Blue™ conjugate (Thermo Fisher Scientific) and propidium iodide (BD Biosciences, Franklin Lakes, NJ, USA) for 15 min at room temperature. Then, after 5-fold dilution with Annexin V-binding buffer, the cells were analyzed using a BD FACSCanto II flow cytometer. Data were processed using the BD FACSDiva 8.0.1 and FlowCal software [54 (link)].