Western blot. Cells (cleaned from FBS) and purified EVs were mixed with 2x Laemmli Sample Buffer and boiled for 5 min at 95C. To a 4-20% Mini-PROTEAN® TGX Stain-Free™ Protein Gel 2 µl of cell (~7 µg) and EV sample (~5.8 ± 1.7 µg) was added and 2.5 µl of all blue precision plus protein standard (ladder) was added. The gel was initially run at a lower speed (20V) for samples to enter the gel and after 15 min the speed was increased to 120V and ran for 45 min in a Mini-PROTEAN® Tetra Vertical Electrophoresis Cell. The protein bands were then transferred to an iBlot™ 2 PVDF membrane in the iBlot™ 2 Gel Transfer Device and the membrane was cut into 5 pieces each one containing cells, EVs and ladder. The membranes were then blocked with either 5% NFDM/TBST (for fibronectin), 5% FBS/TBST (for collagen and CD63) or 0.5% BSA/TBST (for calnexin and CD81) for 45 min. Then the primary antibody was added; anticalnexin (rabbit), anti-collagen (rabbit), anti-CD63 (mouse), anti-CD81 (mouse) and antifibronectin (rabbit) at 1/1000 and incubated over night at 4C (except for anti-fibronectin which was incubated at RT for 1 hour). The next day the samples were washed 5 times with TBST and then secondary antibodies (Goat anti-rabbit igG (H+L)-HRP conjugation or Goat anti-mouse igG (H+L)-HRP conjugation) were added at 1/1000 and incubated for approximately 1 hour. The samples were then washed 5 times with TBST and then washed in only TBS before adding to Clarity™ Western ECL Substrate for 5 min before measuring the chemiluminescence in a ChemiDoc™ imaging system (Bio-Rad).