title: Recovery of urinary nanovesicles from ultracentrifugation supernatants.
abstract: BACKGROUND
  Urinary vesicles represent a newly established source of biological material, widely considered to faithfully represent pathological events in the kidneys and the urogenital epithelium. The majority of currently applied isolation protocols involve cumbersome centrifugation steps to enrich vesicles from urine. To date, the efficiency of these approaches has not been investigated with respect to performing quantitative and qualitative analyses of vesicle populations in the pellet and supernatant (SN) fractions.
  
  
  METHODS
  After the series of differential centrifugations, the final SN was reduced to one-twentieth of the original volume by ammonium sulphate precipitation, with the precipitate pellet subjected to another round of differential centrifugations. Electron microscopy, dynamic light scattering and western blot analysis were used to characterize the vesicles present in individual fractions of interest.
  
  
  RESULTS
  Pellets obtained after the second set of centrifugations at 200 000 g revealed the presence of vesicles which share a common marker profile, but with distinct differences from those seen in the initial 200 000 g pellet used as the reference. This suggests an enrichment of previously uncharacterized urinary vesicles still in solution after the initial centrifugation steps. Analysis of protein yields recovered post-ultracentrifugation revealed an additional 40% of vesicles retained from the SN. Moreover, these structures showed a formidable resistance to harsh treatments (e.g. 95% ammonium sulphate saturation, hypotonic dialysis, 0.3 M sodium hydroxide).
  
  
  CONCLUSIONS
  Methods which employ differential centrifugations of native urine are remarkably ineffective and may lose a substantial population of biologically important vesicle species.
authors.affiliation: Centre for BioAnalytical Sciences, Dublin City University, Dublin, Ireland.
authors.forename: Luca
authors.initials: L
authors.lastname: Musante
authors.forename: Mayank
authors.initials: M
authors.lastname: Saraswat
authors.forename: Alessandra
authors.initials: A
authors.lastname: Ravidà
authors.forename: Barry
authors.initials: B
authors.lastname: Byrne
authors.forename: Harry
authors.initials: H
authors.lastname: Holthofer
mesh_terms: D000328:Adult; D015415:Biomarkers / Q000652:urine*; D015153:Blotting, Western; D022162:Cytoplasmic Vesicles / Q000737:chemistry* / Q000502:physiology*; D005260:Female; D006801:Humans; D008297:Male; D008854:Microscopy, Electron; D014461:Ultracentrifugation; D014556:Urine / Q000737:chemistry*; D055815:Young Adult
chemical_list: D015415:Biomarkers