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Fig. 1 | Journal of Nanobiotechnology

Fig. 1

From: A simple immunoassay for extracellular vesicle liquid biopsy in microliters of non-processed plasma

Fig. 1

Repeated incubations are needed to immuno-capture all available EVs. Melanoma-derived EVs (from Ma-Mel-86c cell line) were enriched by ultracentrifugation. A Nanoparticle tracking analysis (NTA) of melanoma-derived EVs. Average size and concentration of EVs were obtained in a Nanosight equipment capturing three videos of 60 s per measurement, with camera level 12, threshold 10 and temperature of 25 °C. Software NTA 3.1 (Malvern) was used for the analysis. ɸ: diameter. B Transmission Electron Microscopy (TEM). 1 µl EVs were diluted 1:10 in HBS and floated on a carbon-coated 400-mesh 240 Formvar grid, then incubated with 2% uranyl acetate and analysed using a Jeol JEM 1011 electron microscope operating at 245 100 kV with a CCD camera Gatan Erlangshen ES1000W. Pictures were taken at the Electron Microscopy Facility of the CNB. Bar: 200 nm. A representative image is shown. C EV characterization by Western Blot. Whole cell lysates (L) and EVs were loaded in 12% SDS-PAGE gels. Membranes were immunoblotted for detection of: tetraspanins CD9, CD63, CD81 as general EV markers; β-actin as loading control; MICA as cancer-related marker; and calreticulin (CALR) as an endoplasmic reticulum resident protein not present in the EV fraction. Two gels were loaded: one gel, under non-reducing conditions and the other under reducing conditions, for actin detection. One representative experiment out of three is shown. D EV immuno-capture followed by flow cytometry. 3000 anti-CD63-coated beads [or Isotype (IgG) coated as a negative control] were incubated with 3.5 × 106 Ma-Mel-86c derived EVs/μl in 100 μl (3.5 × 108 EVs/tube). Vesicles were detected by flow cytometry after incubation with anti-CD81-PE. Supernatants from the first incubation (SN 24 h), containing unbound vesicles, were incubated again with fresh anti-CD63 beads and EVs captured during this second incubation were analysed by flow cytometry. This procedure was repeated the following 4 days (SN 48 h, SN 72 h, etc., as indicated). The histograms with the Relative Fluorescence Intensity (RFI \(= \frac{{\text{MFI sample}}}{{\text{MFI IgG}}})\) values from a representative experiment out of three is shown. MFI: mean fluorescence intensity

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