TITLE: Human Sample Processing and Isolation of Extracellular Vesicles with Size Exclusion Chromatography
AUTHORS: J. Nathaniel Diehl, Amelia Ray, Lauren B. Collins, Andrew Peterson, Kyle C. Alexander, John S. Ikonomidis, Adam W. Akerman

[DESCRIPTION]
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).”

[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”.

[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:

IZON AFC quick start guide  https://files.izon.com/hubfs/Manuals,%20Technical%20Notes%20and%20Customer%20Support/AFC/AFC%20Quick%20Start%20Guide.pdf
IZON Full Manual  https://f.hubspotusercontent30.net/hubfs/4136435/Manuals,%20Technical%20Notes%20and%20Customer%20Support/AFC/rotational-afc-user-manual-RA1-OQ-001.pdf

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.

[STEPS]
SECTION: Human plasma collection
1. Collect 5 mL peripheral venous blood by a trained nurse or phlebotomist into prelabeled EDTA-coated evacuated tubes.
 

SECTION: Human plasma collection
2. Immediately after the sample is collected, mix the tube thoroughly and store at Room temperature (< 120 min).
SECTION: Human plasma collection
3. Centrifuge whole blood at 2500 x g for 15 min at Room temperature.
SECTION: Human plasma collection
4. Collect the topmost layer (plasma) of supernatant into a 15 mL conical tube.
SECTION: Human plasma collection
5. Centrifuge the plasma again at 2500 x g for 15 min at Room temperature.
SECTION: Human plasma collection
6. Avoiding the bottom ~100 µL of plasma, collect the topmost plasma into a new 15 mL conical tube.
SECTION: Human plasma collection
7. Aliquot desired volumes into labeled freezer-safe microcentrifuge tubes.
 

SECTION: Human plasma collection
8. Snap freeze plasma fractions and store at -81 °C.
SECTION: EV Isolation
9. Thaw human plasma at Room temperature and centrifuge plasma 2500 x g for 15 min at 4 °C.
SECTION: EV Isolation
10. Power on IZON automated fraction collector (AFC).
SECTION: EV Isolation
11. Select SETUP > calibration.
SECTION: EV Isolation
12. Insert a new   qEVsingle column into the column mount and allow the machine to register it.
 

SECTION: EV Isolation
13. Follow on-screen prompts, click OK to proceed to next step.
SECTION: EV Isolation
12.1. Column settings: qEV =  , count = 4, size = 0.2 mL, void = 0.8 mL, sample = 0.15 mL.
SECTION: EV Isolation
15. Click OK to proceed to next step.
 

SECTION: EV Isolation
16. 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.
SECTION: EV Isolation
17. Once the AFC has finished the extraction, combine the first three fractions and discard or store the fourth.
 

SECTION: EV Isolation
18. Discard the used qEVsingle column and clean the central well with a kimwipe delicate task wipe.
 


SECTION: EV Isolation
19. Repeat steps 12 -18 for additional samples.
SECTION: EV Isolation
20. Remove peristaltic pump tubing from below the column mount by removing the plastic cover on the peristaltic pump.
SECTION: EV Isolation
21. Flush plastic tubing with 5 mL filtered water using a 10 mL syringe and replace plastic tubing on AFC.
 

SECTION: EV Isolation
22. 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.

SECTION: EV Isolation
11.1. Follow the on-screen prompts provided by the AFC to calibrate the machine using the provided 10 g weight.
SECTION: EV Isolation
13.1. Permit existing buffer + 2 mL additional 1x sterile filtered PBS to flush the column.
SECTION: EV Isolation
14. Once flush has completed, click OK to proceed to next step.
SECTION: EV Isolation
14.1. Load 150 µL  human plasma sample in a drop-wise manner into the center of the column.
SECTION: EV Isolation
16.1. Continue to add buffer as needed until the extraction is complete.
SECTION: Human plasma collection
4.1. Carefully avoid disruption of the next layer (buffy coat). Leave   of plasma above the buffy coat.
SECTION: EV Isolation
16.2. If the collection stops at this point, ensure adequate buffer remains above the beads in the column and click OK to proceed.