Figure 1

Biophysical characterization of extracellular vesicles (EVs). (A) Negative staining and transmission electron microscopy (TEM) of EVs isolated from all cell lines shows vesicle-shaped particles in a similar size range (size bar – 100 nm). (B) Size distribution analysis by NTA confirms the size range observed by TEM. (C) EV secretion rate evaluation by measuring total particles/mL of initial culture volume. EV-depleted RPMI and EMEM medium were used as negative controls. (D) EV secretion rate evaluation by measuring total particles/10⁶ cells. (E) Correlation between total EVs and total protein (μg) for all cell lines demonstrates a strong linear correlation with an R² = 0.76 (p value < 0.0001). The dotted line represents the 95% confidence interval for the linear correlation. (F) Western blotting confirms the presence and enrichment of EV markers (CD9 and CD81) in EV lysate (4 μg total protein) in comparison to cell lysate (CL) loaded at 4 and 25 μg total protein. On the contrary, GP96 – an endoplasmic reticulum protein is enriched in CL but not EV lysate. (Following transfer, membranes were cut, incubated in respective antibodies, and were imaged using the Licor. Cut, uncropped blots, depicting the full region scanned on the Licor can be found in Supplementary Information Fig. S4).