Fig. 1.

Flotation on iodixanol gradients shows that both large and small EVs have a buoyant density of 1.1 g/ml.A, large and small EVs derived from LN cells were bottom loaded onto iodixanol density gradients. Twelve fractions of 1 ml each were collected from top to bottom from these gradients, and their densities were analyzed by measuring the absorbance at 340 nm. N = 3; result presented as the mean ± SD. B and C, concentration of particles and proteins in the LN iodixanol gradient fractions determined with Nanoparticle Tracking Analysis (B) and Qubit (C), respectively. Data presented as the percentage of the total amount of particles or proteins in fractions 1 to 12. N = 1. D and E, after density flotation and fractionation of LN lEVs and sEVs in high-resolution iodixanol gradients, equal volumes (36 μl; 0.4–8.5 μg protein) of each fraction (fractions 1–8) were loaded onto SDS-PAGE gels. F, representative negative staining electron transmission microscopy images of lEVs and sEVs from the LN high-resolution density fractions (fractions 1–4). Thirty microliters (0.6–2.2 μg protein) were loaded onto the grids per each fraction. The scale bars represent 200 nm. EV, extracellular vesicle; lEV, large extracellular vesicle; sEV, small extracellular vesicle.