Overview
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 and then discard pellet. Filter the recovered supernatant once through 0.22 µm filters.
- During pre-clearing, prepare 25% (w/w) sucrose solution in deuterium oxide by accurately weighing out 1.9 g (± 0.001 g) of sucrose in a universal tube, and then topping up with deuterium oxide until the weight reaches 7.6 g (± 0.001 g).
- Fill up an ultracentrifuge tube (see Table of Materials) with 22.5 mL of pre-cleared CM. Make up the CM to 22.5 mL with 0.22 µm-filtered PBS if the current volume is less than that.
- Place a glass pipette (see Table of Materials) in the tube and, through it, add 3 mL of sucrose solution so that the solution forms a separate layer beneath the CM.
- Carefully place the tube containing layered CM/sucrose solution into the bucket of a swing-out rotor (see Table of Materials) and secure the bucket into the rotor.
- Place the rotor into the ultracentrifuge (see Table of Materials) and spin at 100,000 x g for 1.5 h at 4 °C.
- Collect 2 mL of the sucrose layer and add this to an ultracentrifuge bottle (see Table of Materials) containing 20 mL of filtered PBS for a washing step.
- Place the tubes into a fixed-angle rotor (see Table of Materials) and spin at 100,000 x g for 1.5 h at 4°C.
- Carefully remove the supernatant with a 10 mL serological pipette and resuspend the pellet with 400 µL filtered PBS. Keep this exosome stock at 4 °C or -80 °C for short-term and long-term storage respectively.
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Materials
Name | Company | Catalog Number | Comments |
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. |