title: Human saliva-derived exosomes: comparing methods of isolation.
abstract: ExoQuick-TC(TM) (EQ), a chemical-based agent designed to precipitate exosomes, was calibrated for use on saliva collected from healthy individuals. The morphological and molecular features of the precipitations were compared with those obtained using the classical, physical-based method of ultracentrifugation (UC). Electron microscopy and immunoelectron microscopy with anti-CD63 showed vesicular nanoparticles surrounded by bi-layered membrane, compatible with exosomes in EQ, similar to that observed with UC. Atomic force microscopy highlighted larger, irregularly shaped/aggregated EQ nanoparticles that contrasted with the single, round-shaped UC nanoparticles. ELISA (performed on 0.5 ml of saliva) revealed a tendency for a higher expression of the specific exosomal markers (CD63, CD9, CD81) in EQ than in UC (p>0.05). ELISA for epithelial growth factor receptor, a non-exosomal-related marker, showed a significantly higher concentration in EQ than in UC (p=0.04). Western blotting of equal total-protein concentrations revealed bands of CD63, CD9 and CD81 in both types of preparations, although they were less pronounced in EQ compared with UC. This may be related to a higher fraction of non-exosomal proteins in EQ. In conclusion, EQ is suitable and efficient for precipitation of salivary exosomes from small volumes of saliva; however, EQ tends to be associated with considerably more biological impurities (non-exosomal-related proteins/microvesicles) as compared with UC.
authors.affiliation: Department of Oral Pathology and Oral Medicine, School of Dental Medicine, Tel Aviv University, Tel Aviv, Israel (AZH, DD, MV)
authors.forename: Ayelet
authors.initials: A
authors.lastname: Zlotogorski-Hurvitz
authors.affiliation: Department of Oral Pathology and Oral Medicine, School of Dental Medicine, Tel Aviv University, Tel Aviv, Israel (AZH, DD, MV)
authors.forename: Dan
authors.initials: D
authors.lastname: Dayan
authors.affiliation: Department of Oral and Maxillofacial Surgery, School of Dental Medicine, Tel Aviv University, Tel Aviv, Israel (GC)|Department of Oral and Maxillofacial Surgery, Rabin Medical Center, Petah Tikva, Israel (GC)
authors.forename: Gavriel
authors.initials: G
authors.lastname: Chaushu
authors.affiliation: Departments of Diagnostics and Oral Medicine, Institute of Dentistry, University of Oulu, Finland (JK, TS)|Medical Research Center, Oulu University Hospital, Oulu, Finland (JK, TS)
authors.forename: Johanna
authors.initials: J
authors.lastname: Korvala
authors.affiliation: Departments of Diagnostics and Oral Medicine, Institute of Dentistry, University of Oulu, Finland (JK, TS)|Medical Research Center, Oulu University Hospital, Oulu, Finland (JK, TS)|Institute of Dentistry, University of Helsinki, Helsinki, Finland (TS)
authors.forename: Tuula
authors.initials: T
authors.lastname: Salo
authors.affiliation: Department of Oral Pathology and Oral Medicine, School of Dental Medicine, Tel Aviv University, Tel Aviv, Israel (AZH, DD, MV)|Biocenter Oulu Department of Pathology, Oulu University Hospital, Oulu, Finland (RS)
authors.forename: Raija
authors.initials: R
authors.lastname: Sormunen
authors.affiliation: Department of Oral Pathology and Oral Medicine, School of Dental Medicine, Tel Aviv University, Tel Aviv, Israel (AZH, DD, MV)|Institute of Pathology, The Chaim Sheba Medical Center, Tel Hashomer, Israel (MV).
authors.forename: Marilena
authors.initials: M
authors.lastname: Vered
mesh_terms: D000328:Adult; D000368:Aged; D015153:Blotting, Western; D011232:Chemical Precipitation; D004797:Enzyme-Linked Immunosorbent Assay; D055354:Exosomes / Q000737:chemistry* / Q000648:ultrastructure; D005260:Female; D006801:Humans; D008297:Male; D018625:Microscopy, Atomic Force; D008854:Microscopy, Electron; D008875:Middle Aged; D012463:Saliva / Q000737:chemistry / Q000166:cytology*; D060225:Tetraspanin 28 / Q000032:analysis*; D060245:Tetraspanin 29 / Q000032:analysis*; D060149:Tetraspanin 30 / Q000032:analysis*; D014461:Ultracentrifugation
chemical_list: D060225:Tetraspanin 28; D060245:Tetraspanin 29; D060149:Tetraspanin 30
keywords: ExoQuick; exosomes; extracellular vesicles; isolation; saliva; ultracentrifugation