Evaluation of apoptosis with Annexin V

The influence of Au/ZnO/Ag nanoparticles on the viability of the Jurkat cell line was also assessed by taking into consideration the initiation of apoptosis or necrosis. The apoptotic or necrotic death was evaluated with a FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen) available commercially, in accordance with the procedure specified by the manufacturer. Similarly as in the case of the MTT assay, cell cultures were carried out in the presence of Au/ZnO/Ag nanoparticles at the concentration of 1μM, 10μM, and 100μM for 24, 48, and 72h. The test was conducted in the following manner: first, cells were suspended in 100μl of Annexin buffer (1×), in the amount of 1×10⁵ cells. Then, 5μl of propidium iodide (PI) and 5μl of Annexin V conjugated with fluorescein (FITC) were added to the samples. After incubation in the dark for 15 minutes, 400μl of Annexin buffer (1×) were added to each of the test tubes. Stained samples were acquired with FACS Canto flow cytometer (Becton Dickinson). FACS Diva Software (Becton Dickinson) was used to analyze the results. Based on the proportion between FITC and/or PI fluorescence, cells were defined as early apoptotic, late apoptotic or necrotic [23]. Data were obtained after two independent measurements.