4.6. Annexin V-Propidium Iodide Double Staining Assay

For the detection of apoptosis, the Annexin V binding capacity of treated cells was examined by flow cytometry using an Annexin V-fluorescein isothiocyanate (FITC) Detection Kit (BD Pharmingen, San Jose, CA, USA) according to the manufacturer’s protocol. CT26, HCT116, and SW480 cells were incubated with the appropriate IC₅₀ concentrations (calculated previously by the obtained MTT assay results) of ruthenium(II) terpyridine complexes, Ru-1, Ru-2 and oxaliplatin, or with media alone (control) for 24 h at 37 °C in an atmosphere of 5% CO₂ and at absolute humidity. Following the incubation, all cells were trypsinized, washed in PBS, centrifuged, and resuspended in 100 μL of ice-cold binding buffer (10× binding buffer: 0.1 M Hepes/NaOH (pH 7.4), 1.4 M NaCl, 25 mM CaCl₂) at a concentration of 1 × 10⁶/mL. Annexin V-FITC and propidium iodide (PI) were added to the 100 μL of cell suspension and incubated for 15 min at room temperature (25 °C) in the dark. After incubation, 400 μL of 1× binding buffer was added to each tube and stained cells were analyzed within 1 h using a flow cytometer FACS Calibur (BD Biosciences, San Jose, CA, USA). Data were analyzed using FlowJo Software version VX [45]. Measurements were presented as density plots of Annexin V-FITC and PI stainings.