Fluorescein isothiocyanate fitc conjugated annexin 5

To evaluate cell viability and apoptosis, HMEC-1 and HDF were treated with Cyanoflan at 0.25%, 0.50% and 0.75% (w/v), or left untreated (control), in incomplete cell culture media (without FBS supplement), for 24 h. Then, cells were washed with Phosphate Buffered Saline (PBS) and trypsinized for 5 min at 37 °C. After centrifugation (1200 rpm during 5 min), cell pellets were resuspended in Binding Buffer (Life technologies, Carlsbad, CA, USA). Then, each experimental condition was incubated with 5 µL of Fluorescein isothiocyanate (FITC)-conjugated Annexin V (Life technologies, Carlsbad, CA, USA) and 5 μL propidium iodide (PI, Life technologies, Carlsbad, CA, USA) for 10 min at room temperature protected from light. In addition, the cells were stained only with PI or Annexin V FITC (mono staining) or left unstained (negative control).
Flow cytometry was performed using a BD Accuri C6 Plus Flow Cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA), plotting at least 30,000 events for negative control and mono labels, and 50,000 events for double staining, after debris exclusion. Data was analyzed using FlowJo 10.7.1 software (Tree Star, Inc., Ashland, OR, USA).