Exo fitc dye

Magnetic streptavidin beads were conjugated with tetraspanin-coupled, biotinylated anti-CD9 or anti-CD63 antibodies provided in the Tetraspanin Exo-Flow Combo Capture Kit (System Biosciences, Palo Alto, USA). These magnetic beads were incubated with EV suspension overnight on a rotating mixer at 4 °C. During this step, EVs were captured on to the conjugated magnetic beads. The next morning, magnetic beads were washed thrice in 1X wash buffer to remove any unbound EV particles. EVs captured on to magnetic beads were resuspended in 500 µL wash buffer and incubated with 5 µg of anti-P2X7r antibody conjugated with Alexa Fluor™ 647 overnight on a rotating mixer at 4 °C. The magnetic beads were washed thoroughly to eliminate unbound P2X7r antibodies. EVs were stained with exo-FITC dye (System Biosciences). Cytometric acquisition was performed using an Aurora flow cytometer (Cytek®, San Diego, USA) and analyzed using FlowJo software v10 (Tree Star Inc., Ashland, USA) to check the distribution of P2X7r on EVs.

sEVs were incubated with PKH67 green or PKH26 red fluorescent linker dye (Sigma-Aldrich) (1:500 dilution) in a final volume of 100 μL at RT for 10 min. Reactions were stopped by adding 100 μL of 5% BSA in PBS. sEVs were precipitated using Exoquick-TC solution (System Biosciences) and resuspended in PBS. As described further below, sEVs were also labeled with exo-FITC dye (Systems Biosciences).