29
3. Tris (pH 6.8, 0.5 M).
4. 10% Sodium dodecyl sulfate (SDS, Sigma).
5. 10% Ammonium persulfate (APS, AppliChem), freshly
prepared.
6. Tetramethylethylenediamine (TEMED, Merck).
7. Isopropanol.
8. 2× Laemmli buffer (0.125 mM Tris–HCl, 4% SDS, 20% glycerol, 0.04% bromophenol blue, 10% β-mercaptoethanol,
pH 6.8).
9. 5× Running buffer (125 mM Tris base, 0.96 M glycine, 0.5%
SDS, pH 8.3).
10. 5× Transfer buffer (250 mM Tris base, 192 mM glycine, 0.1%
SDS, pH 9.2).
11. Tris-buffered saline with Tween 20 (TBST) buffer (20 mM
Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20).
12. 0.45 μm Polyvinylidene fluoride membrane (PVDF)
(Immobilon, Millipore).
13. Methanol for hydrophilization of membrane.
14. Blocking buffer (5% nonfat milk powder) in TBS-T.
15. Monoclonal primary antibodies anti-CD63 (Abnova
MAB0931) and anti-CD81 (Abnova MAB6435) in TBS-T
(1:2000).
16. Anti-rabbit IgG secondary antibody in PBS (1:5000) (System
Bioscience).
17. ECL Western Blotting Substrate and SuperSignal West Femto
Substrate (Thermo Scientific) for luminescence-based
imaging.
1. 4% Paraformaldehyde (Santa Cruz).
2. 1% Glutaraldehyde, EM grade (Sigma).
3. 0.5% Uranyl acetate, pH 4.0 (Polysciences).
4. PBS kept at 4 °C.
5. 200 mesh Formvar carbon-coated EM grids (Electron
Microscopy Sciences, FCF300).
6. Parafilm.
7. Clean forceps.
8. Whatman filter paper.
9. 0.22-μm-pore-sized syringe filter.
1. Promega SV total RNA isolation kit.
2. 60% Ethanol.
2.2.3 Transmission
Electron Microscopy
Imaging
2.2.4 RNA Isolation
Ultrasonics-Assisted Effective Isolation and Characterization of Exosomes from Whole…
3. Tris (pH 6.8, 0.5 M).
4. 10% Sodium dodecyl sulfate (SDS, Sigma).
5. 10% Ammonium persulfate (APS, AppliChem), freshly
prepared.
6. Tetramethylethylenediamine (TEMED, Merck).
7. Isopropanol.
8. 2× Laemmli buffer (0.125 mM Tris–HCl, 4% SDS, 20% glycerol, 0.04% bromophenol blue, 10% β-mercaptoethanol,
pH 6.8).
9. 5× Running buffer (125 mM Tris base, 0.96 M glycine, 0.5%
SDS, pH 8.3).
10. 5× Transfer buffer (250 mM Tris base, 192 mM glycine, 0.1%
SDS, pH 9.2).
11. Tris-buffered saline with Tween 20 (TBST) buffer (20 mM
Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20).
12. 0.45 μm Polyvinylidene fluoride membrane (PVDF)
(Immobilon, Millipore).
13. Methanol for hydrophilization of membrane.
14. Blocking buffer (5% nonfat milk powder) in TBS-T.
15. Monoclonal primary antibodies anti-CD63 (Abnova
MAB0931) and anti-CD81 (Abnova MAB6435) in TBS-T
(1:2000).
16. Anti-rabbit IgG secondary antibody in PBS (1:5000) (System
Bioscience).
17. ECL Western Blotting Substrate and SuperSignal West Femto
Substrate (Thermo Scientific) for luminescence-based
imaging.
1. 4% Paraformaldehyde (Santa Cruz).
2. 1% Glutaraldehyde, EM grade (Sigma).
3. 0.5% Uranyl acetate, pH 4.0 (Polysciences).
4. PBS kept at 4 °C.
5. 200 mesh Formvar carbon-coated EM grids (Electron
Microscopy Sciences, FCF300).
6. Parafilm.
7. Clean forceps.
8. Whatman filter paper.
9. 0.22-μm-pore-sized syringe filter.
1. Promega SV total RNA isolation kit.
2. 60% Ethanol.
2.2.3 Transmission
Electron Microscopy
Imaging
2.2.4 RNA Isolation
Ultrasonics-Assisted Effective Isolation and Characterization of Exosomes from Whole…