Materials and Reagents

Burkitt’s Lymphoma B cell lines (e.g. Mutu^-, Mutu I, Mutu III cell lines)
RPMI 1640 medium (Wako Chemicals USA, catalog number: 189-02025 )
Sucrose (Sigma-Aldrich, catalog number: S7903 )
Anti-CD63 monoclonal antibody (clone MEM-250) (Abnova, catalog number: MAB0931 )
Bradford protein assay kit (Bio-Rad Laboratories, catalog number: 500-0006JA )
1, 1'-dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine perchlorate (DiI) (Life Technologies, catalog number: D3911 )
Fetal Bovine Serum (FBS) (Sigma-Aldrich, catalog number: F9423 )
Tris
NaCl
EDTA
Exosome-depleted FBS (see Recipes)
TNE buffer (see Recipes)
0.25-2.5 M sucrose gradient in TNE buffer (see Recipes)

Equipment

10 cm dish
Centrifuge (Eppendorf, model: 5810R or that with equivalent equipment spec)
Ultracentrifuge (Beckman Coulter, model: Optima L-80 XP or that with equivalent equipment spec)
37 °C, 5% CO₂ cell culture incubator
Autopipette
50 ml polypropylene concal plastic tubes (BD Biosciences, Falcon^®, catalog number: 352070 or that with equivalent spec)
SW28 rotor (Beckman Coulter, model: 342204 )
SW40Ti rotor (Beckman Coulter, model: 331301 )
Polyallomer centrifuge tubes 1 x 3½ in (25 x 89 mm) for SW28 rotor (Beckman Coulter, catalog number: 326823 )
Polyallomer centrifuge tubes 9/16 x 3½ in (14 x 89 mm) for SW41Ti rotor (Beckman Coulter, catalog number: 331372 )
Spectrometer
Fluorescent or confocal laser scanning microscope

Procedure

Purification of exosomes
1. Burkitt’s Lymphoma cell lines are grown up from 1 x 10⁷ (in one 10 cm dish) to 2 x 10⁸ cells (in twenty 10 cm dishes) in 200 ml RPMI 1640 medium containing 10% exosome-depleted FBS in the 5% CO₂ incubator at 37 °C.
2. Culture medium containing exosomes are harvested and centrifuged in 50 ml conical tubes at 1,500 rpm for 10 min at room temperature to remove cells.
3. The supernatant is centrifuged in 50 ml conical tubes at 3,500 rpm for 15 min at room temperature to remove cell debris.
4. The supernatant is ultracentrifuged in polyallomer centrifuge tubes at 25,000 rpm for 1 h at 4 °C with an SW28 rotor.
5. The pelleted exosomes are resuspended in 100 µl TNE buffer over night at 4 °C.
6. The exosomes are fractionated by use of a 0.25-2.5 M sucrose gradient in TNE buffer in polyallomer centrifuge tubes at 25,000 rpm for 4 h at 4 °C with an SW40Ti rotor. After that, you will see a band derived from exosomes (if you collect 1 ml of each fraction from the top, exosome fraction usually locates around 6th fraction from the top).
7. The band is collected (about 1 ml) carefully with an autopipette.
8. Fractionated exosomes are ultracentrifuged at 25,000 rpm for 1 h at 4 °C with an SW40Ti rotor.
9. The pelleted exosomes is resuspended in 100 ~ 200 µl TNE buffer over night at 4 °C.
10. The total protein concentration in the fractions is determined by the Bradford protein assay.
11. The fraction containing exosomes (4 µg, each) is confirmed by western blot analysis with anti-CD63 monoclonal antibody (1:1,000 dilution) (Figure 1).
  
  Figure 1. Purified exosomes derived from Burkitt’s lymphoma Mutu cell lines. Exosomes were purified from culture medium of Mutu^- (1^st lane), Mutu I (2^nd lane), and Mutu III (3^rd lane) cells. 4 µg of exosomes were analyzed by western blot with anti-CD63. The arrow indicates the bands that correspond to CD63.
Fluorecent labeling of exosomes
1. 1 ml of fractionated exosomes (100 ng/ml) is incubated with 6 µl of 10 µM stock solution of 1, 1'-Dioctadecyl-3, 3, 3', 3'-Tetramethylindocarbocyanine Perchlorate (DiI) in methanol for 1 h in the dark at room temperature with gentle agitation.
2. Confirm the efficiency of labeling with small amount of exosomes under fluorescent or confocal laser scanning microscope.
3. Aliquot, stored at -80 °C.

Recipes

Exosome-depleted FBS
Ultracentrifuge FBS at 25,000 rpm for 4 h at 4 °C
Collect supernatant and stored at 4 °C
TNE buffer
10 mM Tris-HCl (pH 7.6)
100 mM NaCl
1 mM EDTA
Stored at room temperature
0.25-2.5 M sucrose gradient in TNE buffer
Prepare 0.25 M and 2.5 M sucrose in TNE buffer (Figure 2)
1. Fill 2.5 M sucrose solution up to the half the height of polyallomer centrifuge tubes (approximately 6 ml)
2. Add 0.25 M sucrose solution in layers up to the top of tubes (approximately 6 ml) and plug the tubes with rubber plugs
3. Lay down the tubes with the top slightly higher than bottom (use thin tube floater as a pillow) for al leaset 1.5 h at room temperature
4. Stand the tubes slowly and keep at 4 °C until just before use (storable up to 2 days)
Figure 2. Preparation of 0.25-2.5 M sucrose gradient in TNE buffer. (a) Fill 2.5 M sucrose solution up to the half the height of polyallomer centrifuge tubes (approximately 6 ml). (b) Add 0.25 M sucrose solution in layers up to the top of tubes (approximately 6 ml) and plug the tubes with rubber plugs. (c) Lay down the tubes with the top slightly higher than bottom (use thin tube floater as a pillow) for at least 1.5 h at room temperature. (d) Stand the tubes slowly and keep at 4 °C until just before use (storable up to 2 days).

Acknowledgments

This protocol has been adapted from a previously published paper (Nanbo et al., 2013). This work was supported in part by Grant for Funding from Basic Science research projects from The Sumitomo Foundation; Akiyama Life Science Foundation; Grant-in-Aid for Scientific Research; Shiseido Female Researcher Science Grant; The Sagawa Foundation for Promotion of Cancer Research.

References

Nanbo, A., Kawanishi, E., Yoshida, R. and Yoshiyama, H. (2013). Exosomes derived from Epstein-Barr virus-infected cells are internalized via caveola-dependent endocytosis and promote phenotypic modulation in target cells. J Virol 87(18): 10334-10347.
Nanbo, A., Imai, M., Watanabe, S., Noda, T., Takahashi, K., Neumann, G., Halfmann, P. and Kawaoka, Y. (2010). Ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner. PLoS Pathog 6(9): e1001121.