Figure 6

Evaluating urine-mediated damage of exosomes. Exosomes coupled to microbeads were labelled with a luminal fluorescent dye (Calcein-AM), prior to incubation with various concentrations of NaCl (A) or with fresh urine specimens from four healthy donors (HD1-4) (B). In parallel, identical beads were set up, in the absence of Calcein-AM dye, stained instead with anti-MHC Class-I (RPE) conjugated antibody. After 1 h at room temperature, the fluorescence signal present in the FL-1 channel (Calcein) was compared to FL-2 fluorescence (Class-I-RPE). Graphs show ratio of Calcein to Class I fluorescence. To examine proteolytic damage of exosomes (C), western blot was performed for CD9, TSG101 and PSA on LNCaP-derived exosomes; which were incubated for 2 h or 18 h with fresh urine specimens (from three healthy donors), in the presence or absence of protease inhibitors. Trypsin was used as a positive control for proteolysis.