Fig 1.
Scheme of EV fractionation using density gradient centrifugation.
Large particles in whole saliva such as desquamated epithelia and blood cells were first removed by centrifugation at 2,600 g for 30 min. Then the resultant supernatants were centrifuged at 160,000 g for 70 min to obtain the pellets of particles containing various sizes of EVs. These particles were fractionated by both spinning down (downward, D) and floating up (upward, U) through the density gradient of iodixanol at 160,000 g for 96 h. After centrifugation, ten fractions (F1 to F10) were recovered from the top and analyzed after being concentrated through centrifugation at 160,000 g for 120 min.
Fig 2.
Specimen-specific density drift.
(A) Western blotting for CD63 from specimen 1 (S.1), Specimen 2 (S.2) and Specimen 3 (S.3) both in the downward (D) and upward (U) centrifugation. A position of 48 KDa molecular weight marker is indicated at the right side of S.1 D panel, and the fraction numbers are indicated on the top. (B) The densities of 60 fractions from three specimens (Specimen 1 in blue, 2 in green and 3 in red) both in downward (circles) and in upward (squares) centrifugation are plotted, including those fractions with the strongest signals of CD63 (filled circles and squares).
Fig 3.
Proteomic analyses of the density fractions from three specimens in downward and upward separation.
(A) Venn diagram shows the number of proteins that were detected common to Specimens 1 and 2; the number of proteins that were detected common to Specimens 1 and 3; the number of proteins that were detected common to Specimens 2 and 3; the number of proteins that were detected only in Specimen 1; the number of proteins that were detected only in Specimen 2; and the number of proteins that were detected only in Specimen 3. The blue-colored area represents the number of proteins that were detected only in Specimen 1; the green-colored area, the number of proteins that were detected only in Specimen 2; the red-colored area, the number of proteins that were detected only in Specimen 3, and the pink-colored area, the numbers of proteins that were detected common to Specimens 1 and 2, Specimens 1 and 3, and Specimens 2 and 3, respectively. The size of each circle corresponds to the number of proteins in each category. The list of proteins in each group is provided in S1 Dataset. (B) Graphs showing the densities of the fractions of each specimen in Groups IA-1 to IA-7, IB and some proteins in Group II. The horizontal axis indicates densities that were determined using the refractometer. The circles and squares indicate the fraction numbers having PFD (see text) from the downward and upward direction, respectively. The number of proteins contained in each group is indicated under the name of the group.
Table 1.
List for of proteins classified into IA-1.
Table 2.
List for of proteins classified into IA-2.