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Figure S1. Western blotting for the presence of exosomal markers Alix and TSG101 Western blot analysis indicated absence of Alix and TSG101 in EI and UC exosomal samples from 10 mL of plasma. Colorectal cancer cell line LIM 1863-derived exosomes were used as a positive control.

Figure S2. Comparison of MS based proteomic analysis with the previous studies Venn diagram depicting the protein overlaps of three normal blood plasma exosomal/microvesicle studies. Overall, 304 proteins were identified among which only 20 proteins were common to the three studies.

Figure S3. Characterization of LIM 1863 colorectal cancer cell-derived exosomes (A) Exosome preparations (30 μg) were separated on SDS-PAGE, electrotransferred and probed with antibodies specific against exosome markers Alix and TSG101. (B) Morphology of exosomes was examined by TEM after being negatively stained with uranyl acetate (scale bar, 500 nm) (C) Exosomes spiked and non-spiked plasma sample pellets were separated by SDS-PAGE, transferred to nitrocellulose membrane and probed with an antibody against TSG101. A visible band of TSG101 was detected in the spiked plasma; however, no TSG101 band could be detected in the non-spiked plasma. (D) Western blot analysis of exosomal pellets and concentrates derived from PBS spiked with LIM 1863 exosomes. Pellet A was obtained by the initial differential centrifugation step while Pellet B and C are obtained by two slightly modified exosome isolation protocol using the UC method (Fig. S1). No TSG101 could be detected in the 3 and 100 kDa concentrates revealing that exosomes that are ruptured or unable to settle in the centrifugation protocol are significantly low.

Figure S4. Schematic representation of the protocols used to isolate spiked exosomes from PBS

Figure S5. (A) Western blot analysis of plasma samples spiked with LIM 1863 exosomes and stored at 4°C, -20°C and -80°C. (B) Western blot analysis of plasma samples spiked with LIM 1863 exosomes and stored at 37°C for 10 and 30 days.

Figure S6. Uptake of stored plasma exosomes by LIM 1215 colorectal cancer cells (A) As a control, exosome depleted FCS was mixed with PKH67 dye (scale bar, 50 μm) and incubated with LIM 1215 colorectal cancer cells. (B) Exosomes (-20°C for 3 months) were labelled with the dye PKH67 and incubated with LIM 1215 cells for 4 hours at 37°C. The cells were fixed and analysed by confocal microscopy. The labelled exosomes (green fluorescence) were taken up by LIM 1215 cells (scale bar, 50 μm).

pmic7568-sup-0002-tableS2.xls157KTable S1. List of protein identified by MS using the three isolation methods
pmic7568-sup-0003-supmat.doc54KSupplementary Material

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