Human Sample Processing and Isolation of Extracellular Vesicles with Size Exclusion Chromatography
J. Nathaniel Diehl, Amelia Ray, Lauren B. Collins, Andrew Peterson, Kyle C. Alexander, John S. Ikonomidis, Adam W. Akerman
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).”
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”.
Attachments
Steps
Human plasma collection
Collect 5mL peripheral venous blood by a trained nurse or phlebotomist into prelabeled EDTA-coated evacuated tubes.
Immediately after the sample is collected, mix the tube thoroughly and store at Room temperature (< 2h 0m 0s).
Centrifuge whole blood at 2500x g,0h 0m 0s for 0h 15m 0s at Room temperature.
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 2500x g,0h 0m 0s for 0h 15m 0s at Room temperature.
Avoiding the bottom ~100µL of plasma, collect the topmost plasma into a new 15mL conical tube.
Aliquot desired volumes into labeled freezer-safe microcentrifuge tubes.
Snap freeze plasma fractions and store at -81°C.
EV Isolation
Thaw human plasma at Room temperature and centrifuge plasma 2500x g,0h 0m 0s for 0h 15m 0s at 4°C.
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 10g weight.
Insert a new 35 nm qEVsingle column into the column mount and allow the machine to register it.
Column settings: qEV = 35 nm , count = 4, size = 0.2mL, void = 0.8mL, sample = 0.15mL.
Follow on-screen prompts, click OK to proceed to next step.
Permit existing buffer + 2mL additional 1x sterile filtered PBS to flush the column.
Once flush has completed, click OK to proceed to next step.
Load 150µL human plasma sample in a drop-wise manner into the center of the column.
Click OK to proceed to next step.
Once the sample has completely entered the column, carefully load 2mL 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.
Discard the used qEVsingle column and clean the central well with a kimwipe delicate task wipe.
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 5mL filtered water using a 10 mL syringe and replace plastic tubing on AFC.
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.
