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

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.