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.
The exoEasy Maxi Kit allows isolation of extracellular vesicle from pre-filtered serum, plasma or cell culture supernatant in just 25 minutes. Eluted vesicles are ready for physical or biochemical characterization, or buffer exchange and concentration using e.g., ultrafiltration.
(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.
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.