HEK 293T cells were grown in vesicle-free DMEM for 66 h. Extracellular vesicles (EVs) were isolated using the exoEasy Kit from 15 ml cell culture supernatant pre-filtered using a 0.8 µm filter. Particle concentration was determined using the Nanosight instrument. Isolated EVs were labeled using BODIPY TR Ceramide for 20 min at 37°C. Unincorporated dye was removed by size exclusion chromatography (SEC). As a negative control, an equivalent amount of free dye in PBS was subjected to the same SEC cleanup. To test the uptake of EVs into target cells, approximately 1.5 x 109 labeled particles (representing about 1% of the total eluate from an exoEasy preparation started with 15 ml medium) were incubated with 2 x 104 HEK 293T cells in 200 µl DMEM for 1 h at 37°C. After washing, cells were stained with DAPI to visualize nuclei (bottom row) versus EVs taken up by cells (top row). Left column: negative control, right column: labeled EVs.
(A) HeLa cells were maintained in serum-free medium for 48 h before harvesting culture supernatants. The exoEasy Maxi Kit was used to isolate EVs from 2 ml of supernatant that had been pre-filtered using a 0.8 µm filter. EVs were diluted 1:100 and characterized using the Nanosight NS300 instrument (Malvern, UK). (B) EVs were also isolated from 1 ml of EDTA plasma (measured at 1:200). Mean particle diameters were reported as 212 nm and 214 nm, respectively, which is within the expected range for a mixed population of exosomes and other extracellular vesicles. Red outline indicates variation between 5 repeat measurements.
Scanning electron microscopy (SEM; 20,000x magnification) was performed on a solubilized pellet from ultracentrifugation of pre-filtered (0.8 µm) plasma compared to an exoEasy eluate. Both preparations contain vesicle-shaped structures with an expected size range from 50–200 nm (white arrows; scale bar 200 nm). The preparation from ultracentrifugation also includes many smaller, unidentified structures that do not match the expected shape and size of extracellular vesicles.