Extended Data Fig. 5: EVs bind Axl and are uptaken in lysosomes.
(a) Receptor binding of hybrid EVs. Cy5-labled hybrid semen EVs (shown in red) were added to Vero E6 cells in 8-well ibidi slides at increasing concentrations and slides incubated at 4 °C for 2 h to allow attachment without uptake. Cells were then fixed by addition of 4% PFA and additionally stained for Axl (anti-Axl-PE, Thermo Fisher, #12-1087-42, shown in green) and nuclei (Hoechst, shown in blue). Images were acquired as z-stacks, maximum intensity projections are shown as the pearson’s correlation map, merge and single channels. Mander’s M2 overlap maps were calculated and visualized from z-stacks using Huygens Professional. Images representative of 3 images per concentration. Scale bars = 20 μm. (B-C) Cellular uptake of Cy5-semen EVs on Vero E6 cells. Cells were seeded to an Ibidi chamber one day prior, then stained with LysoTracker DND-26 Green (LysoT, shown in green) before adding Cy5-hybrid EVs (5 × 1010 particles per ml, shown in red) and acquiring z-stacks at indicated time points, the imaging chamber being incubated at standard conditions between time points. Pearson’s correlation coefficient (b) for correlation of Cy5 and LysoTracker channels calculated using LasX software. 6-7 separate z-stacks per time point, means ± SD. Images shown in (c) are representative snapshots of 6-7 total images acquired, scale bars = 20 μm.
