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v. with 1.0��1010 p/g DiR-EVs (left) and PBS-treated control (right). (B) Representative ... To further confirm that we indeed tracked EVs and not only the dye, CD63-EGFP-labelled EVs were injected in mice. HEK293T cells were transfected with a CD63-EGFP plasmid (transfection efficiency of approximately 90%, data not shown) and the subsequent EVs were harvested. According to NTA analysis, approximately 20% of the EVs were EGFP-positive (Supplementary Fig. 2A). Twenty four hours post-i.v. injection of EGFP-positive EVs, the organs were harvested, snap frozen, sectioned and stained with an anti-EGFP antibody. EGFP-positive EVs could be detected in the parenchyma of the liver and spleen (Supplementary Fig. 2B). However, no or only negligible levels of EGFP could be detected Flavoprotein in lungs and kidneys (data not shown). This is probably because the levels of the EVs in other organs are lower and thus below the detection threshold of the technique. Overall, these results corroborate with our results obtained by the DiR-labelled EVs, with the highest accumulation of EVs detected in the liver and spleen. Dose titration of DiR-labelled EVs To investigate whether the dose of EVs injected may affect the biodistribution, a dose-titration study was carried out. All injection doses were determined by using a fixed number of EVs (measured by NTA) per gram body weight of the mice, hence particles/gram body weight (p/g). A positive Rapamycin dose-dependent response of the total tissue fluorescence was observed following i.v. injection of 3 different doses: 0.25��1010, 1.0��1010 and 1.5��1010 p/g of DiR-labelled HEK293T EVs. EVs accumulated mainly in liver, spleen, gastrointestinal tract (GI)-tract and lungs. However, even though an increase in dose resulted in an increased fluorescent signal in the analysed organs (Fig. 2D and E and Supplementary Fig. 3), the relative distribution among the organs shifted. Relative liver accumulation decreased from 77%��2.4 to 60%��3.9 (pABT-263 clinical trial (p
