Odyssey m infrared imager

For in vivo tracking studies, CPC-EVs were labeled with Alexa Fluor 790- or Alexa Fluor 647 NHS ester dyes (Thermo Fisher Scientific). EVs were incubated with 0.05 nM reactive dye in 0.1 M NaHCO₃ in phosphate-buffered saline (PBS) and incubated for 30 min at 37°C while shaking at 450 rpm. After labeling, the free amine-reactive dye was quenched using a final concentration of 0.1 M Tris-HCl for 30 min at RT. Quenched free dye was removed using a Sepharose CL-4B column coupled to an ÄKTA start system (GE Healthcare) containing a UV 280 nm flow cell. EV-containing fractions were concentrated using a 100 kDa MWCO Amicon Ultra-4 spin filter (Merck Millipore). Fluorescent labeling efficiency was determined by diluting different EV volumes in 50 µl PBS and measuring fluorescence at 800 nm using an Odyssey M Infrared Imager (LI-COR Biosciences).

At termination, individual organs were collected, snap-frozen and stored at −80°C. Organs were weight, cut, and lysed in 1x RIPA buffer (Abcam) using ceramic beads and a Precellys 24 tissue homogenizer (Bertin Instruments). Total fluorescence of 50 µl lysed sample was measured using an Odyssey M Infrared Imager (LI-COR Biosciences) at 800 nm. Fluorescent units were determined as mean grey value, corrected for background fluorescence of organs collected from the negative control (PBS-injected) mice.