This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
Enrichment of extracellular vesicles using Millipore membrane filters
Louise C. Laurent
University of California San Diego, Sanford Consortium for Regenerative Medicine, 2880 Torrey Pines Scenic Drive, La Jolla, CA
Corresponding Author
Roger P. Alexander
Sample and Assay Standards Working Group of the Extracellular RNA Communication Consortium
Corresponding Author
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
This protocol describes how to enrich extracellular vesicles from cell culture using Millipore membrane filters in order to detect, identify and quantify extracellular RNA.
Keywords
exRNA, extracellular vesicles, exosomes, Millipore
Figure 1
Extracellular RNAs (exRNAs) have been identified in every biofluid that has been tested. They have been found in extracellular vesicles, ribonucleoprotein complexes and lipoprotein complexes. exRNAs are interesting because they may serve as signalling molecules between cells, they have the potential to serve as biomarkers for prediction and diagnosis of disease, and exRNAs or the extracellular particles that carry them might be used for therapeutic purposes.
The Sample and Assay Standards Working Group of the Extracellular RNA Communication Consortium (ERCC) is a group of laboratories funded by the U.S. National Institutes of Health to develop robust and standardized methods for collecting and processing of biofluids, separating different types of exRNA-containing particles and isolating and analyzing exRNAs. In our first joint endeavour, we held a series of conference calls and in-person meetings to survey the methods used among our members, placed them in the context of the current literature and used our findings to identify areas in which the identification of robust methodologies would promote rapid advancements in the exRNA field.
A full list of the protocols developed during this effort is available at the exRNA Portal, the ERCC's website ( "http://exrna.org/resources/protocols/":http://exrna.org/resources/protocols/ ). This protocol for enrichment of extracellular vesicles from cell culture using Millipore membrane filters is one of the extracellular vesicle and particle enrichment methods compared in "the associated publication":http://www.journalofextracellularvesicles.net/index.php/jev/article/view/26533.
phosphate-buffered saline (PBS), pH 7.4
Steriflip, 0.22 μm PES membrane (EMD Millipore, catalog # SCGP00525)
FalconTM 50mL Conical Centrifuge Tubes, polypropylene (Fisher, catalog # 14-432-22)
AU-15 filter, 15 mL, 10 kDa MWCO (EMD Millipore, catalog # UFC901024)
AU-0.5 filter, 0.5 mL, 10kDa MWCO (EMD Millipore, catalog # UFC501024)
Microfuge
Microfuge tubes, 1.5 mL
Table 1. Equipment list in tabular format
For cell culture supernatant:
- Transfer supernatant media1 from cell culture flask to a 50 mL conical tube.
- Screw the Steriflip unit onto the top, open end of the sample tube. Secure tightly.
- Invert the assembly and secure in a tube base or rack.
- 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.
- Detach the Steriflip device and cap sample. Store filtered sample at 4°C or proceed with microvesicles enrichment.
- Add 2 mL PBS to the AU-15 filter, cap and centrifuge for 10 minutes at 4000 xg minutes in a swinging bucket rotor.
- Remove unfiltered PBS from the bottom of the filter device. Aspirate filtrate from the collection tube.
- Add 15 mL sample to the AU-15 filter and cap the device.
- Centrifuge at for 30 minutes at 4000 xg. There should be ~500 μl sample remaining in the upper chamber.
- Remove device from the centrifuge and empty the collection tube.
- Add 14 mL PBS to the filter device and gently pipette sample multiple times to mix.
- Centrifuge at for 30 minutes at 4000 xg.
Recover concentrated sample from the upper chamber of the filter device.
For serum or plasma:
- Add 500 μl PBS to the AU-0.5 filter, cap, and centrifuge for 10 minutes at 14,000 xg.
- Reverse the device and centrifuge for 2 minutes at 1000 xg to remove the residual PBS.
- Aspirate PBS from the collection tube.
- Transfer 200 μl sample and 300 μl PBS to the AU-0.5 filter and cap the device.
- Centrifuge for 30 minutes at 14,000 xg. There should be ~15 μl sample remaining in the upper chamber.
- Remove device from the centrifuge and empty the collection tube.
- Add 0.5 mL PBS to the filter device and gently pipette sample multiple times to mix.
- Centrifuge for 30 minutes at 14,000 xg.
- Place filter upside down in a fresh microcentrifuge tube.
- Centrifuge for 2 minutes at 2000 xg to transfer the sample to the tube.
1) Use of serum-free media is recommended to avoid concentration of overly abundant albumin that is present in Fetal Bovine Serum (FBS). Exosome-free media, such as Exo-FBSTM Exosome depleted media (System Biosciences), will still contain albumin. If albumin is not a concern, the protocol may be used on serum-containing samples. To achieve similar concentration, centrifugation times must be lengthened due to increased protein content.
Version History
CURRENT STATUS: Posted
Version 1
Posted 21 Dec, 2015
Metrics
Comments: 0
HTML Views: ...
Associated Publications
Meeting report: discussions and preliminary findings on extracellular RNA measurement methods from laboratories in the NIH Extracellular RNA Communication Consortium
by Louise C. Laurent, Asim B. Abdel-Mageed, P. David Adelson, +12
Method Article
Enrichment of extracellular vesicles using Millipore membrane filters
Louise C. Laurent
University of California San Diego, Sanford Consortium for Regenerative Medicine, 2880 Torrey Pines Scenic Drive, La Jolla, CA
Corresponding Author
Roger P. Alexander
Sample and Assay Standards Working Group of the Extracellular RNA Communication Consortium
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 21 Dec, 2015
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
This protocol describes how to enrich extracellular vesicles from cell culture using Millipore membrane filters in order to detect, identify and quantify extracellular RNA.
Figure 1
Extracellular RNAs (exRNAs) have been identified in every biofluid that has been tested. They have been found in extracellular vesicles, ribonucleoprotein complexes and lipoprotein complexes. exRNAs are interesting because they may serve as signalling molecules between cells, they have the potential to serve as biomarkers for prediction and diagnosis of disease, and exRNAs or the extracellular particles that carry them might be used for therapeutic purposes.
The Sample and Assay Standards Working Group of the Extracellular RNA Communication Consortium (ERCC) is a group of laboratories funded by the U.S. National Institutes of Health to develop robust and standardized methods for collecting and processing of biofluids, separating different types of exRNA-containing particles and isolating and analyzing exRNAs. In our first joint endeavour, we held a series of conference calls and in-person meetings to survey the methods used among our members, placed them in the context of the current literature and used our findings to identify areas in which the identification of robust methodologies would promote rapid advancements in the exRNA field.
A full list of the protocols developed during this effort is available at the exRNA Portal, the ERCC's website ( "http://exrna.org/resources/protocols/":http://exrna.org/resources/protocols/ ). This protocol for enrichment of extracellular vesicles from cell culture using Millipore membrane filters is one of the extracellular vesicle and particle enrichment methods compared in "the associated publication":http://www.journalofextracellularvesicles.net/index.php/jev/article/view/26533.
phosphate-buffered saline (PBS), pH 7.4
Steriflip, 0.22 μm PES membrane (EMD Millipore, catalog # SCGP00525)
FalconTM 50mL Conical Centrifuge Tubes, polypropylene (Fisher, catalog # 14-432-22)
AU-15 filter, 15 mL, 10 kDa MWCO (EMD Millipore, catalog # UFC901024)
AU-0.5 filter, 0.5 mL, 10kDa MWCO (EMD Millipore, catalog # UFC501024)
Microfuge
Microfuge tubes, 1.5 mL
Table 1. Equipment list in tabular format
For cell culture supernatant:
- Transfer supernatant media1 from cell culture flask to a 50 mL conical tube.
- Screw the Steriflip unit onto the top, open end of the sample tube. Secure tightly.
- Invert the assembly and secure in a tube base or rack.
- 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.
- Detach the Steriflip device and cap sample. Store filtered sample at 4°C or proceed with microvesicles enrichment.
- Add 2 mL PBS to the AU-15 filter, cap and centrifuge for 10 minutes at 4000 xg minutes in a swinging bucket rotor.
- Remove unfiltered PBS from the bottom of the filter device. Aspirate filtrate from the collection tube.
- Add 15 mL sample to the AU-15 filter and cap the device.
- Centrifuge at for 30 minutes at 4000 xg. There should be ~500 μl sample remaining in the upper chamber.
- Remove device from the centrifuge and empty the collection tube.
- Add 14 mL PBS to the filter device and gently pipette sample multiple times to mix.
- Centrifuge at for 30 minutes at 4000 xg.
Recover concentrated sample from the upper chamber of the filter device.
For serum or plasma:
- Add 500 μl PBS to the AU-0.5 filter, cap, and centrifuge for 10 minutes at 14,000 xg.
- Reverse the device and centrifuge for 2 minutes at 1000 xg to remove the residual PBS.
- Aspirate PBS from the collection tube.
- Transfer 200 μl sample and 300 μl PBS to the AU-0.5 filter and cap the device.
- Centrifuge for 30 minutes at 14,000 xg. There should be ~15 μl sample remaining in the upper chamber.
- Remove device from the centrifuge and empty the collection tube.
- Add 0.5 mL PBS to the filter device and gently pipette sample multiple times to mix.
- Centrifuge for 30 minutes at 14,000 xg.
- Place filter upside down in a fresh microcentrifuge tube.
- Centrifuge for 2 minutes at 2000 xg to transfer the sample to the tube.
1) Use of serum-free media is recommended to avoid concentration of overly abundant albumin that is present in Fetal Bovine Serum (FBS). Exosome-free media, such as Exo-FBSTM Exosome depleted media (System Biosciences), will still contain albumin. If albumin is not a concern, the protocol may be used on serum-containing samples. To achieve similar concentration, centrifugation times must be lengthened due to increased protein content.
Associated Publications
Meeting report: discussions and preliminary findings on extracellular RNA measurement methods from laboratories in the NIH Extracellular RNA Communication Consortium
by Louise C. Laurent, Asim B. Abdel-Mageed, P. David Adelson, +12