Visualization of Mutual EV Uptake

To visualize the mutual EV uptake, cells were first dyed with fluorescent, lipophilic carbocyanine tracers, green DiO (ThermoFisher Scientific, Waltham, MA, USA, catalog #D3898), and red DiI (ThermoFisher Scientific, catalog #D3886). Right before seeding, cells were treated with 1 uM dye followed by incubation at 37 °C for 5 min [33 (link)]. The GBM was dyed red with DiI, whereas the NSCs were dyed green with DiO. EVs secreted by each cell line take the color of the dye of the parent cell. GBM and NSCs were independently cultured for 24 h in a 6-well non-tissue culture treated plate in 4 mL of culture media, and the cells were incubated at 37 °C with 5% CO₂. The next day, the cells and the conditioned media from each cell line were centrifuged at 0.3 rcf for 5 min at room temperature. The GBM cell pellet was resuspended in NSC-conditioned media, whereas the NSC pellet was resuspended in the GBM-conditioned media with further incubation under the same conditions. The controls did not receive the media switch. After 48 h, the cells were ready to be fixed and visualized using confocal microscopy.