Fig. 1
Chromatographic separation of EV-containing fractions present in cell culture media. Proteins present in SKBR3 cell culture supernatant (a) and in the kit-purified EV-enriched fraction from the same supernatant (b) were separated using a large-pore anion-exchange monolith column. Three and four independent elution fractions were detected, respectively, and fractions corresponding to peaks 1 and 3 (black and red bars) eluted at coincident salt concentrations in the two samples. All the three fractions separated from the cell-culture supernatant sample were analyzed by flow-cytometry and resulted positive for the EV marker CD9 (c). The irrelevant anti-mouse PE-labeled antibody was used to evaluate possible unspecific interactions between EV fractions and antibodies during flow-cytometry. Gates were set according to the values of autofluorescence of naked beads—not coated with EVs