Tracking Extracellular Vesicle Uptake

s.END1 EC-EVs were labeled with C. elegans cel-miR-39-3p transcript (Exiqon) using an exosomal transfection kit (Exo-Fect, System Bioscience) according to the manufacture’s instructions. EC-EVs were then washed by ultracentrifuging at 120,000 g for 120 minutes in 13 ml PBS. Labeled EC-EVs were injected into mice by tail vein injection at a concentration of 1 × 10⁹ EVs, and tissues of interest harvested. Tissues were perfused with heparinized PBS (10 ml) through insertion of a 25-gauge needle into the left ventricle. Tissues and plasma were harvested and snap frozen for qPCR analysis; harvested tissues were mechanically homogenized. To determine the uptake of labeled EVs by resident splenic cell populations (splenic monocytes vs. splenic ECs) isolated spleens were dissociated and splenocytes were incubated with anti-CD31 –conjugated (clone MEC 13.3, BD Pharmingen) magnetic beads (sheep anti-rat IgG Dynabeads, Invitrogen) to isolate splenic ECs as previously described (70 (link)). The resultant supernatant containing unbound cells was enriched for monocyte populations using EasySep (Stemcell Technologies) as previously described (1 (link)). Isolated cells were lysed and prepared for miRNA RT-qPCR.