Supplementary Figure 8: Formalin–EDC fixation of cultured human embryonic kidney 293 (HEK293) cells permit imaging of extracellular vesicles (EVs) near the cell surface with transmission electron microscopy (TEM).

a, Western blot of cell lysates and isolated extracellular vesicles (EV iso) from HEK293 cells, probed for TSG101, CD81, CD9, or Syntenin 1 to verify for enrichment within EV preparation. Samples were tested for GM130 and cytochrome C as negative markers. Beta actin antibodies were used as a control. b, Nanoparticle tracking analysis (NTA) graphical representation of the size and concentration of extracellular vesicles (EVs) isolated from the culture media from HEK293 cells. The mean (black line) ± standard error (red bars) concentration of EVs according to EV diameter, based on NTA shows EVs are present in HEK293 media. c–e, Representative TEM photomicrographs of a formalin–EDC-fixed HEK293 tissue sections show EV-shaped electron dense signal in the extracellular space (closed arrowheads) near the cell surface in three biological replicates. (c) The photomicrographs from HEK293 replicate no. 1 show an EV near the cell body, and the inset (black box) depict the EV (arrowhead) at higher magnification (right). d, Representative TEM photographs from HEK293 replicate no. 2 show an outpouching of EV-like structure (bottom inset) connected to a cell membrane (middle, single arrowhead). The top inset shows an EV (black box) at the external to the cell (right, double arrowhead). e, A third biological replicate shows EV-like structures adjacent to the cell membrane (arrowheads). Scale bars are (c) 500 nm (left) and 100 nm (right), (d) 500 nm (left), 250 nm (middle) and 200 nm (right), and (e) 500 nm (left) and 200 nm (right).