Fig. 3

Cationic polymer addition increased EV detection by flow cytometry and ELISA. Lung cancer-derived EVs (from H3122 cell line) and melanoma-derived EVs (from Ma-Mel-86c cell line) were enriched by ultracentrifugation. 1 × 10⁶ H3122 EVs/µl or 1.8 × 10⁷ Ma-Mel-86c EVs/µl in 100 µl of PBS-Casein 1% were treated with 4 or 8 μg/ml polybrene (PB), 4 or 8 μg/ml poly-l-lysine (PL) or kept untreated for 18 h, as indicated in each panel. A Flow Cytometry. EVs were incubated with 3000 of either anti-EpCAM, anti-MICA, anti-CD63 or IgG isotype control beads. Captured vesicles were detected by flow cytometry after incubation with anti-CD81-PE. Histograms from one representative experiment out of 3 are shown in the top panels. Relative Fluorescence Intensity (\({\text{RFI}} = \frac{MFI sample}{{MFI IgG}})\) values are shown in each plot. Bar plots in the bottom panels represent the change in the RFI relative to the untreated condition. Mean and Standard Deviation from three independent experiments are represented. MFI: Median Fluorescence Intensity. B Titration of cationic polymers. Tissue culture supernatant-derived EVs from the lung cancer cell line H3122 were incubated with either 3000 anti-EpCAM or IgG isotype control coated beads in 100 μl final volume of PBS-Casein 1%. EpCAM-captured vesicles were detected by flow cytometry after incubation with anti-CD81-PE. Relative increase of the RFI for EpCAM-CD81 detection, obtained in five experiment replicates (EXP), is shown. Statistical analysis was performed by a Two-way ANOVA Fisher’s LSD test. (*p < 0.05). C ELISA. EVs enriched from tissue culture supernatant of the indicated cell lines were incubated in anti-EpCAM, anti-MICA or anti-CD63 antibody-coated plates. IgG Isotype-coated wells were used as negative control. Detection was performed with biotinylated anti-CD9 antibody followed by SA-HRP. Optical Density (OD) was measured at 450 nm. After subtraction of the negative control, binding relative to the untreated sample is represented. Statistics from three independent experiments are shown: one-way ANOVA Fisher’s LSD (*p < 0.05; 95% confidence interval). A single representative experiment is shown in Additional file 1: Figure S6