Filtration through Amicon® filter. For further purification and concentration, a 10kDa Amicon® filter was used. 15 mL portions of the sample were centrifuged at 1450-3000 RCF for 40 min (Eppendorf Centrifuge 5810 R). To collect the EVs, the filter was rinsed several times with the concentrate and the bottom of the filter was scratched with a 200 µL pipette tip. Afterwards, 200 µL of DPBS was added and the procedure was repeated. The collected EVs were stored in an Eppendorf Biopur® in the fridge to avoid degradation and 10 µL was put aside for NTA measurements. Surface epitope labelling of the EVs with the MACSPlex EV Kit. Labelling of the EVs was performed according to the MACSPlex EV kit instructions. In short, 6.43•10 10 particles (EVs) were used in 120 µL total volume (diluted with MACSPlex buffer). Only MACSPlex buffer was used as blank. The MACSPlex EV capture beads were vortexed and 15 µL were added to both the sample and the blank. The sample and blank were then incubated overnight at room temperature, protected from light using a VWR® tube rotator on a permanent run (18 rpm). The next day 500 µL of buffer was added and centrifuged at 3000 RCF (High speed Microcentrifuge CT15RE, VWR Hitachi) for 5 min. 500 µL of the supernatant was then aspirated. 5 μL of each MACSPlex EV detection reagent CD9, CD63, and CD81 was added to each tube and mixed by pipetting up and down. The samples were incubated for 1 hour at room temperature protected from light using a VWR® Tube Rotator on permanent run (18 rpm). Then 500µL of buffer was added and the samples were centrifuged at 3000 RCF for 5 min (High speed Microcentrifuge CT15RE, VWR Hitachi) followed by aspiration of 500 µL of the supernatant. An additional 500 µL of buffer was added and incubated for 15 min protected from light using a VWR® tube rotator on permanent run (18rpm) followed by centrifugation at 3000 RCF for 5 minutes (High speed Microcentrifuge CT15RE, VWR Hitachi) and aspiration of 500 µl of the supernatant. The sample was then measured using FACS. achieve a concentration in the range for the assay. Then 150 µL of each standard and sample was pipetted into a 96-well plate and an equal amount of WR was added. The well plate was then placed on an orbital shaker (KS 260 basic, IKA) for 30 seconds to mix the samples, followed by incubation at 37°C for 2 h. The well plate was then cooled to room temperature and the absorbance at 562 nm was measured using a Tecan Microplate Reader Spark. The absorbance of the BSA standards was then plotted against the BSA concentration to get a standard curve and a standard curve equation. The concentration of the EV sample could then be calculated from the standard curve equation.