View and FACs labeled exosomes captured on magnetic beads

Exosomes from HEK293 cells grown in Exo-FBS exosome-depleted media supplement with standard DMEM were isolated using ExoQuick-TC. The exosome pellet was resuspended in 1 ml 1x PBS and contained an exosome protein content of 1 ug/ul. About 500 ul of this exosome suspension was labeled with 50 ul of either 10x Exo-Red or Exo-Green for 10 minutes at 37°C. The exosomes were re-isolated using the addition of 100 ul ExoQuick-TC (included in kits) and precipitation at 5°C for 30 minutes on ice or 4°C. The labeled exosome pellets were resuspended in 500 ul 1x PBS. CD63-coupled magnetic beads from SBI's Exo-Flow IP kit (cat# EXOFLOW32A-CD63). Briefly, 50 ul of CD63 magnetic beads were incubated with 100 ul of the labeled exosomes overnight at 4°C on a rotator. The following day, the bead/labeled exosomes were placed on a magnetic plate for 5 minutes and then washed with 100 ul 1x wash buffer once. Then, 100 ul 1x PBS was added to the IP well and placed on the magnetic rack for 2 minutes to position the beads at the bottom of the wells. The beads with captured, labeled exosomes were imaged using fluorescent microscopy.