Exosome Extraction and Purification Kit (Milk)

Additional Information

Exosome Extraction and Purification Procedure

Sample Pretreatment

1. Sample preparation: For frozen sample: take out from the fridge and thaw in 25 °C water bath, then place the fully-thawed sample on ice; For fresh sample, collect and immediately place the sample on ice.
2. Initial dosage of the sample: The sample volume for a single extraction is recommended to be no less than 20 mL of cell culture supernatant.
3. Centrifugation to remove cell debris: Transfer the sample to the centrifugal tube and centrifuge at 3,000×g (~5,200 rpm **) for 10 min at 4 ℃ to remove the cell debris in the sample (Note: For significant sediment, repeat centrifugation at 3,000 × g for 10 min until no obvious sediment remains, collecting the supernatant each time).  **Converted by a large centrifuge with an effective centrifugal radius of approximately 10 cm (≥15 mL centrifugal tube), the same applies below.
4. Centrifugation to remove impurity debris: Transfer the centrifugal supernatant to a new centrifugal tube and centrifuge at 10,000 × g (~9,500 rpm**) for 10 min at 4°C to remove impurity debris from the sample.
5. Supernatant transfer: Transfer debris-free supernatant to new centrifugal tube.

Exosome Extraction

1. Supernatant pretreatment: Add Exosome Concentration Solution (ECS reagent) to the supernatant after centrifugation and filtration.
2. Solution mixing: After adding ECS reagent, tightly cover the centrifugal tube and vortex mix for 1 min, then place it at 4℃ for at least 8 h; (Note: Extending the standing time can enhance exosome yield, but should not exceed 24 hours.
3. Precipitation of exosome: Take out the centrifugal tube with the mixed solution and centrifuge at 4℃ at 10,000 ×g（~9,500 rpm**）for 60 min. Discard the supernatant, and the sediment is rich in exosome particles(Note: Aspirate the supernatant as much as possible.
4. Re-centrifugation: The centrifugal tube containing the sediment is centrifuged again at 10,000 × g (~9,500 rpm**) for 2 min at 4°C, and the supernatant is discarded in order to remove any residual liquid from the wall of the tube (Note: Aspirate the supernatant as much as possible.
5. Resuspension of exosome: Resuspend the centrifugal sediment with an appropriate amount of 1× PBS by gently pipetting.） After complete dissolution, transfer the resuspended solution into a new 1.5 mL centrifugal tube(Note: It is recommended that each 20 mL of cell culture supernatant be resuspended with about 200 μL of 1× PBS.
6. Exosome particles harvesting: Centrifuge the 1.5 mL centrifugal tube containing the resuspended solution at 12,000 × g (~12,400 rpm*) for 2 min at 4°C and collect the supernatant, which is rich in exosome particles (Note: If significant sediment persists, repeat centrifugation at 12,000 × g for 2 min until no obvious sediment remains, collecting the supernatant each time.

Exosome Purification

1. Purification of exosome: Transfer the harvested crude exosome particles into the upper chamber of the Exosome Purification Filter (EPF column) and centrifuge at 3,000 × g (~6,200 rpm*) for 10 min at 4°C. After centrifugation, collect the liquid at the bottom of the EPF column tube, which is the purified exosome particles (Note: the EPF column cannot be reused).
2. Preservation of exosome: Aliquot the purified exosomes into appropriate volumes and store at -80℃ in a cryogenic refrigerator for subsequent experiments.