Exocap composite kit

When cells seeded in a 60 mm dish reached 75% confluency (~2.5 × 10⁶ cells), the supernatant media were collected and pre-cleared of cell debris by centrifugation at 2500 rpm for 10 min at 4 °C. Exosomes were isolated from the pre-cleared supernatant culture media of cells using ExoQuick TC reagent (System Biosciences, EXOTC50A-1, Palo Alto, CA, USA), according to manufacturer’s instructions. Briefly, 1 mL of reagent was added per 5 mL of culture media, and incubated at 4 °C for at least 12 h. Exosomes present in the incubated media were then pelleted down by centrifugation at 1500× g for 30 min and resuspended in either 50 µL of PBS or TRIzol reagent (Sigma, St. Louis, MO, USA) for RNA isolation or in RIPA protein lysis buffer for Western blot, and stored in −80 °C until further use. Serum exosomes were isolated from 250 µL serum using ExoCap composite kit (MBL International, Woburn, MA, USA) per instruction manual, which is based on an antibody coupled magnetic capture bead-based procedure. The kit contains a mixture of CD9, CD63, CD81 and EpCAM capture beads. This step was followed by the purification of exosomal RNA using ExoCap Nucleic acid elution buffer (MBL International, MEX-E kit, Woburn, MA, USA), according to the kit protocol.