For measuring the TNHs, ZnO NCs or EVs internalization in KB cells, 3 × 104 cells were cultured into a 24-well plate (Corning, 24-well TC-treated microplates) with 500 μl of complete cell culture medium for 24 h. Then, cell medium was replaced with freshly prepared solutions containing 5, 15, 25 μg/ml of TNHs (labeled with both DiO for EVs and Atto647 for the ZnO NCs) or, as control samples, with 5, 15, 25 μg/ml of ZnO NCs (labeled with Atto647) or with DiO-labeled EVs at the same concentration used for the preparation of 15 μg/ml of TNHs. Untreated KB cells were also prepared as reference. After 24 h, cells were washed with PBS, trypsinized, centrifuged at 130 × g for 5 min, and then resuspended in 500-μl PBS for the cytofluorimetric analysis. 1 × 104 events were collected with a Guava Easycyte 6-2 L flow cytometer (Merck Millipore, MA, USA), with 0.59 μl/s flow rate, excluding cell debris. The analyses were performed by using the blue laser (λex = 488 nm) to detect EVs and the red one (λex = 642 nm) for ZnO NCs.
The positive events were characterized by a shift of Red-R fluorescence intensity (emission filter 661/15) for Atto647-ZnO NCs signal and a shift of Green-B fluorescence intensity (emission filter 525/30) for DiO-EVs signal. The percentages of positive events were compared with untreated cells, evaluated with Guava InCyte Software (Merck Millipore).
The positive events were characterized by a shift of Red-R fluorescence intensity (emission filter 661/15) for Atto647-ZnO NCs signal and a shift of Green-B fluorescence intensity (emission filter 525/30) for DiO-EVs signal. The percentages of positive events were compared with untreated cells, evaluated with Guava InCyte Software (Merck Millipore).