Annexin 5 fluorescein isothiocyanate fitc detection kit

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 (link)]. Measurements were presented as density plots of Annexin V-FITC and PI stainings.

For the apoptosis assay, Annexin V–fluorescein isothiocyanate (FITC) detection kit (BD Biosciences, Erembodegem, Belgium) was used according to the manufacturer’s introduction. The cells were collected, washed twice with PBS, resuspended in Annexin V–FITC and propidium iodide (PI), and stained in the dark for 15 min at room temperature [29 (link)]. Subsequently, the cell apoptosis rate was analyzed using flow cytometry (fluorescence-activated cell sorting, BD Biosciences). For cell cycle distribution analysis, the cells were harvested and fixed in 70% ice-cold ethanol overnight. Then, the cells were centrifuged and resuspended in PBS containing PI (BD Biosciences) and RNase (100 μg/mL) as well as Triton X-100 (0.2%) for 30 min. Finally, flow cytometry was used to analyze cell cycle distribution [29 (link)]. The tests were performed three times for each sample.