Cryo-electron microscopy imaging of trophectoderm sEVs was performed as described. [36] Particle size distribution trophectoderm sEVs was determined by nanoparticle tracking analysis (NanoSight NS300, Malvern), as previously described. [32, 49] 2.5. Lipophilic Dye Labelling of Small Extracellular Vesicles and Uptake Assay L2-TSC cells grown to 80% confluency were labelled with fluorescent dye, DiO (Invitrogen) at 1 μM concentration for 15 min at 37°C as described. [47] Labelled L2-TSCs were washed three times with PBS.
Fresh medium was added, cells incubated for 48 h, after which DiO-labelled Tsc-sEVs were collected at 100 000 × g (1 h). Ishikawa cells were grown to 70% confluency in 8-well glass chamber slides (Sarstedt). Cells were incubated with DiO-labelled sEVs at 37°C for 2 h. Cells were then washed twice in PBS. Nuclei were stained for 10 min with Hoechst 33342 stain (Thermo Fisher Scientific) (10 μg/ml) prior to imaging by Nikon A1R confocal microscope equipped with resonant scanner, using a 40x WI (1.15 NA); (Nikon, Tokyo, Japan). Images were sequentially acquired. The dXY image resolution was 0.21 µm and Z-interval of 0.5 µm. Cell were maintained at 37°C, 5% CO2.
Fresh medium was added, cells incubated for 48 h, after which DiO-labelled Tsc-sEVs were collected at 100 000 × g (1 h). Ishikawa cells were grown to 70% confluency in 8-well glass chamber slides (Sarstedt). Cells were incubated with DiO-labelled sEVs at 37°C for 2 h. Cells were then washed twice in PBS. Nuclei were stained for 10 min with Hoechst 33342 stain (Thermo Fisher Scientific) (10 μg/ml) prior to imaging by Nikon A1R confocal microscope equipped with resonant scanner, using a 40x WI (1.15 NA); (Nikon, Tokyo, Japan). Images were sequentially acquired. The dXY image resolution was 0.21 µm and Z-interval of 0.5 µm. Cell were maintained at 37°C, 5% CO2.