Figure 1
Enrichment and characterization of milk EVs. (a) Protein quantification of milk EVs enriched using (1) crude ultracentrifugation (20.48 µg/µl) and (2) ultracentrifugation with sucrose gradient purification (0.21 µg/µl). After ultracentrifugation with sucrose gradient, enriched milk EVs were pooled (n = 3) and characterised by: (b) Nanoparticle tracking analysis to demonstrate distribution of particle size and their concentration; (c) Flow cytometry to detect the milk EV-associated surface marker CD63 where the X-axis indicates fluorescence intensity linked to CD63, while the Y-axis plots fluorescence of propidium iodide relating to the presence of non-viable cells which are not expected after EV isolation procedures ; (d) Transmission electron microscopy to assess morphology, showing milk EVs at 62,000 × magnification (scale bar 500 nm) and a single milk EV at 100,000 × magnification (scale bar 200 nm); (e) Full length immunoblots showing positive EV-membrane surface markers CD81, CD63 and CD9, and negative EV-markers calnexin and histone 3. Lanes indicated with negative symbol (−) are negative controls and positive symbol (+) indicate EV protein loading. Figures in panel a and b were generated with GraphPad Prism version 6.0.0 for Windows (www.graphpad.com); image for panel c was exported from FlowJo (www.flowjo.com).
