Figure 1

EV isolation protocol flow chart and validation of the technique. (a) EV isolation flow chart. MDA-MB-231 are grown until a density of ±80% is reached. Subsequently, cells are washed and the culture medium is replaced by its serum-free counterpart. On days 2 and 3, the conditioned medium (CM) is harvested, centrifuged and filtered to remove dead cells, large vesicles and debris. Next, the CM is concentrated to <500 µl using ultrafiltration and loaded on top of a 11.5 ml discontinuous OptiPrep gradient. Following 18 h ultracentrifugation, 1 ml fractions are harvested and centrifuged separately to pellet the EVs. Finally, EVs are resuspended in PBS and if desired, stored at −80 °C. (b) Validation of the isolation protocol by Western blot. EVs in the CM are separated according to their density after ODG centrifugation and are harvested in 12 discrete fractions. A representative Western blot of these fractions confirms the presence of EV-markers CD63, TSG101, flotillin-2 and CD9 mainly in fractions 6 and 7 (EV+ fractions). All fractions were clear from contaminating cell organelles as indicated by the absence of markers of the Golgi apparatus (golgin-245), endoplasmic reticulum (calnexin), peroxisomes (PMP70) and mitochondria (cytochrome c). CL = crude lysate. For reasons of clarity and conciseness, blots were cropped to the bands of interest. Samples derive from the same experiment and gels/blots were processed in parallel. Full-length blots are presented in Supplementary Figure S10. (c) Validation of the isolation protocol by shotgun MS. Venn diagram of proteins detected by MS in EV+ fractions 6 and 7 versus exosome markers listed in the ExoCarta Top 100 (published on the ExoCarta database http://exocarta.org/). 1,217 different proteins were detected in the EV+ fractions (light grey plus white section), of which 95 proteins (white intersection) also occur in the ExoCarta Top 100 list. Serum albumin and α-2-macroglobulin (dark grey section) were absent in MDA-MB-231 EV+ fractions but are most likely contaminating proteins. Additional to the proteins detected by Western blot, EV-markers flotillin-1, HSP90-α, HSP70, CD82, CD81 and ALIX were identified in the EV+ fractions (light grey and white section).