Sep 21, 2022
Human Sample Processing and Isolation of Extracellular Vesicles with Size Exclusion Chromatography
Peer-reviewed method
- J. Nathaniel Diehl1,
- Amelia Ray1,
- Lauren B. Collins1,
- Andrew Peterson2,3,
- Kyle C. Alexander2,3,
- John S. Ikonomidis2,3,
- Adam W. Akerman2,3
- 1University of North Carolina School of Medicine, Chapel Hill, North Carolina;
- 2Department of Surgery, University of North Carolina – Chapel Hill, Chapel Hill, North Carolina;
- 3Division of Cardiothoracic Surgery, University of North Carolina – Chapel Hill, Chapel Hill, North Carolina
External link: https://doi.org/10.1371/journal.pone.0284875
Protocol Citation: J. Nathaniel Diehl, Amelia Ray, Lauren B. Collins, Andrew Peterson, Kyle C. Alexander, John S. Ikonomidis, Adam W. Akerman 2022. Human Sample Processing and Isolation of Extracellular Vesicles with Size Exclusion Chromatography. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4jeb2lo5/v1
Manuscript citation:
Diehl JN, Ray A, Collins LB, Peterson A, Alexander KC, et al. (2023)A standardized method for plasma extracellular vesicle isolation and size distribution analysis. PLOS ONE 18(4): e0284875. https://doi.org/10.1371/journal.pone.0284875
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 26, 2022
Last Modified: September 21, 2022
Protocol Integer ID: 69217
Keywords: Extracellular vesicles, EVs, Exosomes, TRPS, Tunable resistive pulse sensing, Human plasma, Automatic fraction collector, AFC, qEV, Size exclusion chromatography, qNano Gold,
Abstract
This protocol details the steps necessary to isolate circulating plasma extracellular vesicles (EVs) from human peripheral blood samples. This protocol utilizes an automated fraction collector (AFC) and qEV size-exclusion chromatography columns from IZON Science. This is intended to serve as the first step in the workflow for EV quantification along with “Measurement of extracellular vesicles with tunable resistance pulse sensing (TRPS).”
Attachments
ix3ebwxfp.docx
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Guidelines
- All human blood samples should be handled according to the Centers for Disease Control (CDC) universal blood and body fluid collection guidelines. Additionally, sample handling and processing should follow the Occupational Safety and Health Administration (OSHA) blood borne pathogens procedures to prevent possible pathogen transmission.
- This protocol details the extraction of EVs from human peripheral blood samples. All human subjects research should be reviewed and approved by the site-specific Institutional Review Board (IRB) prior to proceeding.
- qEVsingle columns are used in this protocol. Newer generation multi-use columns can also be used if column-specific instructions are followed.
Citations:
Additional Notes:
- If the AFC continuously stops running in the middle of an extraction and it is not due to running out of buffer, check that the tubing is not obstructed by removing it from the tubing apparatus and confirming that liquid can be pushed through. Tubing can be replaced if necessary.
- For further troubleshooting or information, please refer to the IZON AFC Quick Start Guide and the IZON AFC Full Manual.
Materials
- Sterile alcohol prep padsFisher ScientificCatalog #22-363-750
- 25-gauge Safety-Lok needle and collection setBdCatalog #367285
- BD Vacutainer EDTA tubeFisher ScientificCatalog #23-021-013
- Cryogenic tubes with screw capFisher ScientificCatalog #03-337-7Y
- Microcentrifuge tubesEppendorfCatalog #022364111
- qEVsingle 35 nm Legacy column, IZON, PC#SP6
- 200 μL filtered pipet tipsMillipore SigmaCatalog #CLS4823-960EA
- 1000 μL filtered pipet tipsMillipore SigmaCatalog #CLS4809-1000EA
- 10 mL Sterile syringe with Luer lock, Fisher Scientific, Cat#14-955-460
- 0.22 μm Luer lock inlet filtersThomas ScientificCatalog #1176G49
- PBS TabletsThermo FisherCatalog #18912014
- Filtered (0.22 µm ) deionized water
Equipment:
- Ultra-low temperature (-80 °C ) freezer
- Standard manual defrost laboratory (-20 °C ) freezer
- Refrigerated microcentrifugeEppendorfCatalog #022620700
- Clinical Centrifuge, Globe Scientific, Item# GCC-E
- qEV Automatic fraction collector (AFC)
- 20 µL -200 µL pipette
- 100 µL - 1000 µL pipette
Safety warnings
Appropriate personal protective equipment (PPE) should be worn (nitrile gloves, safety goggles, and lab coat). Store all organic solvents in a flammable storage cabinet in accordance with institution policy. Utilize biohazard waste containers for sample waste.
Before start
- Ensure that you have an adequate quantity of buffer (1x sterile-filtered PBS)
- If you already have frozen human plasma samples, skip to “EV Isolation”.
Human plasma collection
Human plasma collection
Collect 5 mL peripheral venous blood by a trained nurse or phlebotomist into prelabeled EDTA-coated evacuated tubes.
Note
EDTA anticoagulant is recommended for isolation of EVs.
Immediately after the sample is collected, mix the tube thoroughly and store at Room temperature (< 02:00:00 ).
2h
Centrifuge whole blood at 2500 x g for 00:15:00 at Room temperature .
15m
Collect the topmost layer (plasma) of supernatant into a 15 mL conical tube.
Carefully avoid disruption of the next layer (buffy coat). Leave 1 cm of plasma above the buffy coat.
Centrifuge the plasma again at 2500 x g for 00:15:00 at Room temperature .
15m
Avoiding the bottom ~100 µL of plasma, collect the topmost plasma into a new 15 mL conical tube.
Aliquot desired volumes into labeled freezer-safe microcentrifuge tubes.
Note
We recommend a minimum aliquot volume of 250 µL .
Snap freeze plasma fractions and store at -81 °C .
EV Isolation
EV Isolation
15m
15m
Thaw human plasma at Room temperature and centrifuge plasma 2500 x g for 00:15:00 at 4 °C .
15m
Power on IZON automated fraction collector (AFC).
Select SETUP > calibration.
Follow the on-screen prompts provided by the AFC to calibrate the machine using the provided 10 g weight.
Insert a new 35 nm qEVsingle column into the column mount and allow the machine to register it.
Note
The display will indicate specific column features once it has registered.
Column settings: qEV = 35 nm , count = 4, size = 0.2 mL , void = 0.8 mL , sample = 0.15 mL .
Follow on-screen prompts, click OK to proceed to next step.
Permit existing buffer + 2 mL additional 1x sterile filtered PBS to flush the column.
Once flush has completed, click OK to proceed to next step.
Load 150 µL human plasma sample in a drop-wise manner into the center of the column.
Click OK to proceed to next step.
Note
AFC should move from flush position to collect column buffer into the central well.
Once the sample has completely entered the column, carefully load 2 mL 1x PBS into the top of the column in a drop-wise manner.
Continue to add buffer as needed until the extraction is complete.
If the collection stops at this point, ensure adequate buffer remains above the beads in the column and click OK to proceed.
Once the AFC has finished the extraction, combine the first three fractions and discard or store the fourth.
Note
The fourth fraction contains primarily protein and a very low concentration of EVs.
Discard the used qEVsingle column and clean the central well with a kimwipe delicate task wipe.
Note
Ensure the central well is completely dry before next use.
Repeat steps 12 -18 for additional samples.
Remove peristaltic pump tubing from below the column mount by removing the plastic cover on the peristaltic pump.
Flush plastic tubing with 5 mL filtered water using a 10 mL syringe and replace plastic tubing on AFC.
Note
This will prevent buildup of salt crystals in the tubing between uses.
Combine fractions 1-3 and immediately process for use on TRPS, store short term (1-2 weeks) at -20 °C , or store long term (> 2 weeks) at -81 °C .