Fig. 4

Isolation and characterization of PS-positive EVs. (a) Flowchart of the PS-EVs isolation process. (b) Cryo-electron microscopy of non-collapsed extracellular vesicles (left), demonstrating the native character of isolated vesicles, with an apparently intact double membrane. The EVs cryo-TEM micrographs were collected on Falcon3 direct electron detection camera at the 120,000 × nominal magnification with the under focus in the range 3–10 µm and the overall dose of < 20 e/Å2; scale bar equals 50 nm. Negative staining transmission electron micrograph of cup-shaped extracellular vesicles (right). Shown is a heterogeneous population of vesicles consisting of a range of sizes (30–200 nm) with low densities typical for exosomes. The extracellular vesicles were isolated by MagCapture isolation kit and embedded in a mixture of 2% uranyl-acetate. EVs were examined at 70 kV with a Morgagni 268D transmission electron microscope. Both Cryo-electron and TEM microscopy were performed on a fresh sample of EVs not subjected to freezing temperatures; scale bar equals 500 nm. (c) Size of isolated extracellular vesicles determined by DLS (Dynamic Light Scattering). (d) MagCapture (PS) pp = PS-EVs proteins characterized with a sufficient number of proteotypic peptides; MagCapture (PS) topEV = PS-EVs proteins identified in this study and present in the Top 100 proteins list of often identified in EVs according to EV databases ExoCarta and Vesiclepedia