Exo flow ip kit

Isolated exosomes from serum or cell supernatants were suspended in 1 ml of 1X PBS. Exosome aliquots (500 μl) were labeled with 50 μl of 10X Exo-Red [SBI] according to manufacturer’s instructions. The exosomes were re-isolated using the addition of 100 μl ExoQuick followed by precipitation for 30 min at +4 °C. The labeled exosome pellet was suspended in 500 μl 1X PBS and stained with CD63-coupled magnetic beads provided by SBI’s Exo-Flow IP kit [SBI] and with the FITC-conjugated peptides.

For the validation of macrophage polarization, the antibody, anti-mouse CD86 (FITC) (cat. no. 11–0860-82, eBioscience), was incubated with cells for 45 min at 4 °C and washed three times with fresh PBS to remove the excess.
The happened liposomes-EVs hybridization was validated in order to check the co-localization of Rhodamine-DHPE lipid and anti-CD63-APC. Specifically, vesicles were conjugated with CD63-coupled magnetic beads provided by SBI’s Exo-Flow IP kit (SBI) according to the manufacturer’s instructions. Immunoconjugates were then stained with anti-CD63-APC (cat. No. 130–108-894, Miltenyi Biotec). The markers’ expression was then quantified through FACSCanto II flowcytometer (Becton Dickinson, San Jose, CA, USA) and analyzed with FlowJo software (Tree Star, Inc., Ashland, OR, USA).