Exosome Block Preparation: A Protocol to Preserve Exosomes From Cultured Human Colon Cancer Cells

Abstract

Source: Jung, M. K. et al. Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy. J. Vis. Exp. (2018)

This video describes the technique of preparing exosome blocks from cultured gastrointestinal tumor cells. These exosome blocks can further be sliced, stained, and imaged to determine the position of specific proteins in the lipid bilayer vesicle.

Protocol

1. Block Preparation of Exosomes

1. Pellet the exosomes from the culture supernatant of HCT116 cells by centrifugation at 100,000 x g for 1.5 h. Remove the culture supernatant and carefully fix the purified exosome pellet with 1 mL of 2.5% glutaraldehyde in 0.1 M sodium cacodylate solution (pH 7.0) for 1 h at 4 °C. For 0.1 M sodium cacodylate, dissolve 4.28 g cacodylic acid in 160 mL of distilled water (DW). Adjust pH to 7.4 with 0.1 M HCl, then make up to 200 mL with distilled water.
2. Remove the fixative and rinse the pellets with 1 mL of 0.1 M sodium cacodylate buffer at room temperature. Repeat three times, with each change lasting 10 min.
3. Post-fix the sample with 1 mL of 2% osmium tetroxide for 1 h at 4 °C.
4. Remove the fixative and rinse three times with 0.1 M sodium cacodylate buffer every 10 min.
5. Incubate for 10 min with a graded acetone series (50%, 60%, 70%, 80%, 90%, 95%, 100%, respectively) on the shaker.
6. Remove the acetone and incubate the solution of 3:1 acetone:low viscosity embedding mixture for 30 min. The exosome pellet is in the tube.
7. Remove the medium and add 1:1 acetone:low viscosity embedding mixture medium, then incubate for 30 min.
8. Remove the medium and add 1:3 acetone:low viscosity embedding mixture medium, then incubate for 30 min.
9. Remove the medium and add 100% low viscosity embedding mixture and incubate overnight at room temperature.
10. Embed the sample in pure low viscosity embedding mixture using the embedding mold and bake for 24 h at 65 °C.

Materials

Glutaraldehyde	EMS	16200
Sodium cacodylate	EMS	12300
Osmium tetroxide 1 g crystal	EMS	19100	Use only in fume hood
Acetone	JUNSEI	1B2031