Red fluorescent cell linker for general cell membrane

Lyophilized urinary CD63, CD9, CD81 positive EVs (HansaBioMed, Estonia) were diluted in ultra-pure water to a protein concentration of 100 µg/mL, following manufacturer instructions. Prior to staining, 1.2 µL of 1 mM PKH26 stock (Red Fluorescent Cell linker for General Cell Membrane, Sigma-Aldrich) was added to 300 µL of diluent C and incubated at 37 °C for 15 minutes. Then, 1 µL of EVs stock was added to PKH26 in diluent C, resulting in a sample with final concentrations of 0.3 µg/mL of EVs and 4 µM of PKH26. Other concentrations of EVs (4.5, 1.5 and 0.03 µg/mL) and PKH (20 and 0.16 µM) were made following the same overall procedure in diluent C.

ARPE-19 cell culture medium was collected for sEVs isolation. sEVs were obtained by differential ultracentrifugation. To label sEVs, 1 mL of Diluent C and 4 mL of PKH26 (Red Fluorescent Cell linker for General Cell Membrane, Sigma-Aldrich, St. Louis, MO, USA) were added to the sEVs pellet, followed by 1 h of incubation. The staining process was stopped by adding an equal volume of DPBS. Labeled sEVs were then subjected to ultracentrifugation at 120,000× g for 1 h and resuspended in exo-free serum cell media. HUVEC were cultured on a 24 mm circular coverslip up to 80% confluence, then mounted into the recording chamber. sEVs labeled with PKH26 from ARPE-19 medium were added to HUVEC. Pictures were acquired using a Leica TCS SP8 Inverted Confocal Microscope (Leica, Wetzlard, Germany) with 63X oil immersion objective and a Leica DM IL LED inverted microscope. The settings for detecting the PKH26 emission fluorescence were excitation at 550 nm and emission at 667 nm. Fluorescence quantification was performed using grayscale with the LasX software Office 1.4.5.