Figure 2
Isolation of exosomes using the REIUS method. (a) Physical characteristics of HADF and melanoma cell line exosomes based on NTA (Zetaview). Exosome polydispersity index (NePdi) is the ratio of standard deviation over mean exosome size based on NTA (Zetaview). (b) Scanning electron microscope images of HADF exosomes and six HMEX. REIUS method isolated exosomes consistently resemble circular vesicles in shape. (c) Infrared-based western blot staining with exosome-enriched marker anti-CD63 and cytoplasmic marker anti-β-tubulin antibodies. Lane 1: 25 µg HMEX protein following REIUS method using a 100 kDa ultrafiltration spin column. Lane 2: 25 µl flow-through following SEC of REIUS method. Absence of CD63 expression indicates no loss of exosomes following entire REIUS method. Lanes 3 and 4: same as lanes 1 and 2 except that a 3 kDa ultrafiltration spin column was used. Similar intensity of CD63 expression in lanes 1 and 3 indicate exosomes are not lost during the REIUS method when a 100 kDa spin column is used. Lanes 5 and 6: same as lanes 1 and 2 but with HADF exosomes. Lane 7: 25 µl HMEX supernatant following REIUS that did not include SEC. The increased lane width indicates presence of non-exosomal protein that would have been removed if SEC had been utilized. Lane 8: 2183-Her4 cell lysate. β-tubulin expression is only seen in this lane, indicating preparations in other lanes are not contaminated by cellular protein. (d) HADF exosomes and HMEX from 6 cell lines: infrared-based western blot staining with anti-CD63, anti-TSG101, anti-β-tubulin and anti-β-actin antibodies. 25 μg protein was loaded per lane. Full-length blots for are presented in Supplementary Figs S2 and S3.
