FIGURE 7.

Characterization of sEVs collected with different strategies. sEVs were isolated from αMEM+8% PL (M) (1 and 5; with stripes) in order to verify the presence of PL-derived sEVs in the media. MSCs were grown in αMEM+8% PL and αMEM+8% EV depl. PL. sEVs were collected for 24 h (2 and 6) and 48 h (3 and 7) for αMEM+8% PL cultures and for 48 h for αMEM+8% EV depl. PL cultures (4 and 8). Methods I (blue scheme) and IV (orange scheme) were used for sEV isolation from M and CM. (A) Western blotting was performed in order to investigate the expression of proteins GRP94, Flot-1, CD63, ApoA1, and CD81. MSC cell lysate (CL) and EV depl. PL served as controls for primary antibodies, and ß-actin was used as loading control. Reducing conditions for indicated proteins were obtained by the addition of dithiothreitol (DTT). (B) Chemiluminescent signal intensities were quantified and normalized on ß-actin intensities, resulting in ß-actin-normalized expression of proteins. (C) sEVs were visualized by negative contrast staining using TEM with 60,000 times magnification. The black scale bar represents 500 nm. (D) + (E) Mean and modal size of sEVs was determined by NTA. Representative images are depicted for western blotting and TEM. Data are presented as mean ± SD, and N ≥ 4 independent experiments were performed for NTA analyses. Statistically significant differences are depicted as follows: *: p < 0.05, **: p < 0.01.