Exosome Marker Detection and Purification

Purification of SE was further verified by expression of the exosome markers, CD63 and CD91 (link)22 (link). Detection of human CD63 and human CD9 in SE using flow cytometry was completed per exosome-human CD63 isolation/detection kit following manufacturer’s instructions (Invitrogen). Briefly, 25 μg of SE re-suspended in isolation buffer (0.1% BSA in PBS) were incubated with 20 μl of 4.5 μm-diameter magnetic polystyrene beads (Dynabeads) at 4 °C overnight with rotation. The Dynabeads are pre-coated by the manufacturer with a primary monoclonal antibody for human CD63 antigen. The SE-bound beads were then washed three times (0.1% BSA in PBS) to remove unbound exosomes, and the exosome-bound beads were stained for flow cytometry with anti-CD63-FITC (Biolegend) or anti-CD9-PE (Biolegend) for 1 hr at RT in the dark on an oscillating mixer. The SE-bound beads were then washed three times (0.1% BSA in PBS) to remove unbound antibody. CD63 or CD9 levels were analyzed through a FACSVerse instrument and mean fluorescence intensity was determined using FlowJo software (Tree Star). Further details are provided in the Supplementary Material in accordance with MIFlowCyt standards23 (link).