Extracellular Vesicle Protocol

Enrich for extracellular vesicles in just 40-60 minutes using centrifugal filters. If you are discouraged by ultracentrifugation or precipitation methods for preparing exosomes and other extracellular vesicles (EVs), try this rapid protocol that outperforms exosome precipitation methods.

Enrichment of the Microvesicle Fraction from Cell Culture Supernatant Using Amicon^® Ultra Centrifugal Filters

This protocol that follows was designed for enrichment of extracellular vesicles (EVs) from serum-free cell culture media. The process involves sample pre-clarification (to remove cells and cell debris) by vacuum-based filtration using a Steriflip^® device. The filtered sample is then concentrated by centrifugation using an Amicon^® Ultra (AU) filter (10k MWCO). A second spin is performed for sample cleanup and buffer exchange via centrifugal diafiltration. The final concentrated sample is ready for downstream analysis or application.

Note: All four Amicon^® Ultra filter sizes (0.5, 2, 4 and 15 mL) are compatible with the workflow; please consult the each user guide for specific information regarding appropriate centrifuge/rotors and spin speed maxima.

Note: We recommend using serum-free media to avoid concentration of overly abundant albumin that is present in Fetal Bovine Serum (FBS). If albumin is not a concern, the protocol may be used on serum-containing samples. To achieve similar concentration, centrifugation times must be lengthened (Steps 9 and 11, below) due to increased protein content.

Note: FBS also contains exosomes, which may interfere with certain downstream analyses. Exosome-free media, such as Exo-FBS™ Exosome depleted media (System Biosciences), will still contain albumin.

Step 1. Sample Clarification

1. Transfer supernatant media from cell culture flask to a 50 mL threaded conical tube.
2. Screw the Steriflip^® unit [# SCGP00525; 0.22 µm Millipore Express (PES) membrane] into the top, open end of the sample tube. Secure tightly.
3. Invert the assembly and secure in a tube base or rack.
4. Attach a regulated vacuum source to the vacuum port on the side of the Steriflip^® unit. Turn on the vacuum to draw solution through the membrane and into the empty tube.
5. Detach the Steriflip^® device and cap sample. Store filtered sample at 4°C or proceed with vesicle enrichment.

Centrifugal ultrafiltration protocol outlined is based on processing 15 mL sample (maximum volume) using an AU-15 filter (10 kDa MWCO, # UFC901024). Please consult the appropriate Amicon^® Ultra filter user guide if different volumes or device formats (AU-0.5, -2, or -4 mL) are to be used.

Step 2. Device Equilibration

6. Add 2 mL PBS to the device, cap and centrifuge at 4,000 x g for 10 minutes in a swinging bucket rotor.

• For AU-2 and 4, the same centrifuge speed can be used. If a fixed angle rotor is to be used, please consult the device user guide for appropriate speed and volume constraints.
• For AU-0.5, samples are spun at 14,000 x g in a fixed rotor that can accommodate 1.5 mL tubes.

7. Remove unfiltered PBS from the bottom of the filter device. Aspirate filtrate from the collection tube.

• For AU-2, 4, and 15 devices, remove filtrate by inserting a pipetter (p200) into the bottom of the filter device and withdrawing sample using a side to side sweeping motion to ensure complete removal.
• For AU-0.5 devices, remove filtrate by reverse spin at 1,000 x g for 2 minutes. Aspirate PBS from collection tube or attach a clean microcentrifuge tube prior to proceeding.

Step 3. Sample Ultrafiltration (Concentration and Buffer Exchange)

8. Add 15 mL sample to the AU-15 filter and cap the device.
9. Centrifuge at 4,000 x g for 30 minutes.
   NOTE: On average, this will yield a final sample of 500 µL (30-fold concentration). However, depending on the nature of the sample, flow rate, and therefore centrifugation time required to achieve the desired concentration, can vary. For estimates on processing lesser volumes, please consult the individual Amicon^® Ultra filter user guides.
10. Remove device from the centrifuge and empty the collection tube.
11. Add 14 mL PBS to the filter device and gently pipette sample multiple times. Centrifuge at 4,000 x g for 30 minutes.
12. Recover concentrated sample from the filter device.

• For AU-2, -4, and-15 devices, insert a pipettor (p200), withdraw a volume of sample (200 µL), and wash down the membrane side walls multiple times (3-4 on each side) to ensure complete recovery. Recover sample volume from base of filter device
• For AU-0.5, place the filter upside down in a clean microcentrifuge tube. Centrifuge at 2,000x g for 1 minute to transfer sample to the tube (reverse spin).

13. The extracellular vesicle sample is ready for analysis.