Exosome Isolation: A Density-based Ultracentrifugation Technique Using Sucrose Cushion to Separate Exosomes from Conditioned Media of Cultured Cells
Published: April 30, 2023
Abstract
Source: Faruqu, F. N. et al. Preparation of Exosomes for siRNA Delivery to Cancer Cells. J. Vis. Exp. (2018)
This video describes a density-based ultracentrifugation technique for isolating exosomes from conditioned media harvested from human embryonic kidney cell cultures using a 25% (w/w) sucrose cushion prepared in deuterium oxide. The isolated exosomes can be used for downstream applications.
Protocol
1. Exosome Isolation onto a Sucrose Cushion Pre-clear the harvested conditioned media (CM) by differential centrifugation and filtration as follows. Centrifuge at 500 x g for 5 min at 4 °C. Transfer the supernatant into a new tube and discard the pellet. Repeat this centrifugation step once more, recovering the supernatant and discarding the pellet. Centrifuge the supernatant from step 1.1. at 2,000 x g for 15 min and 4 °C an…
Disclosures
The authors have nothing to disclose.
Materials
| Sucrose | Fisher Scientific | S/8600/60 | 1 kg |
| Deuterium oxide (D2O) | Sigma-Aldrich | 151882 | 250 g |
| 1X Phosphate Buffered Saline | Thermo Fisher Scientific | 10010015 | 500 ml |
| Millex-GP Syringe Filter Unit 0.22 µm | Millipore | SLGP033RS | |
| Ultracentrifuge | Beckman Coulter | Optima XPN-80 | |
| Swing-out rotor | Beckman Coulter | SW45 Ti | |
| Fixed-angle rotor | Beckman Coulter | Type 70 Ti | |
| Ultracentrifuge tubes | Beckman Coulter | 355631 | Polycarbonate. Max. fill 32 ml |
| Ultracentrifuge bottles | Beckman Coulter | 355618 | Polycarbonate. Min. fill 16 ml, max. fill 25 ml |
| Centrifuge | Eppendorf | 5810R | |
| Glass pipettes | Fisher Scientific | 1156-6963 | |
| 25% w/w sucrose cushion | Add 1.9 g (± 0.001 g) of sucrose in a universal tube, and top up with D2O until the weight reaches 7.6 g (± 0.001 g). This makes ~6ml of the 25% w/w sucrose cushion. |
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Cite This Article
Exosome Isolation: A Density-based Ultracentrifugation Technique Using Sucrose Cushion to Separate Exosomes from Conditioned Media of Cultured Cells. J. Vis. Exp. (Pending Publication), e20565, doi: (2023).