IZON qEV2 Extra Cellular Vesicles User Manual

IZON qEV2 Extra Cellular Vesicles

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Izon Science Ltd PO Box 9292 Addington Christchurch 8024 New Zealand
Telephone: +64 3 357 4270
Email: support@izon.com
Website: www.izon.com

DEFINITIONS AND WRITING CONVENTIONS

Make sure to follow the precautionary statements presented in this guide. Safety and other special notices will appear in boxes and include the symbols detailed in Table 1.
Table 1: Safety and Hazard Symbols

This symbol indicates general advice on how to improve procedures or recommends measures to take in specific situations.
This symbol indicates where special care should be taken.

Table 2: Terminology Used in this Manual

Buffer volume

(Previously void volume)

| The volume of liquid that corresponds to the volume before the Purified Collection Volume (PCV); this volume is collected

automatically into the central well. The buffer volume is different for each column type.

Chromatography| A method used primarily for separation of the components of a sample. The components are distributed between two phases; one is stationary while the other one is mobile. The stationary phase is either a solid, a solid supported liquid, or a gel. The stationary phase may be packed in a column, spread as a layer or distributed as a film. The mobile phase may be gaseous or liquid.
Column volume| The combined volume of packed material and buffer volume (can be referred to as the bed volume).
Degassing| Degassing involves subjecting a solution to vacuum to “boil” off excess dissolved gas e.g. applying a vacuum to a flask.
Flow rate| The volumetric flow in mL/min of the carrier liquid.
Purified Collection Volume (PCV)| The volume immediately succeeding the buffer volume, containing particles of interest purified from the loaded sample. The PCV is different for each column type.
Recovery rate| The percentage of vesicles that come out of the column compared with what went in.
Smart Column| A qEV column with an integrated RFID chip. This chip is used by the AFC instrument to recognize the type of column attached to the instrument. This chip does not affect manual usage of the qEV column.

SAFETY AND HAZARDS

Refer to the Safety Data Sheet for the classification and labelling of hazards and associated hazard and precautionary statements. The Safety Data Sheet for qEV SMART columns is located at
izon.com/sds

Hazards

qEV SMART columns are a laboratory product. However, if biohazardous samples are present, adhere to current Good Laboratory Practices (cGLP) and comply with any local guidelines specific to your laboratory and location.

Disposal of Biohazardous Material
The qEV SMART column contains < 0.1% sodium azide, which is potentially fatal if swallowed or in contact with skin. Please review the following guidelines and precautions prior to each use of the qEV SMART column:

Prevention:

1. Do not get into eyes, on skin, or on clothing.
2. Wash skin thoroughly after handling.
3. Do not eat, drink, or smoke when using this product.
4. Avoid release of product into the environment.
5. Wear protective gloves and clothing; follow general laboratory precautions.

Response

1. IF SWALLOWED: immediately call a POISON CONTROL CENTRE/Doctor.
2. IF ON SKIN: Gently wash with plenty of soap and water and immediately call a POISON CONTROL CENTRE/Doctor.
3. Remove immediately any contaminated clothing and wash before reuse.
4. Collect any spillage and dispose of appropriately.

For more information, see the MSDS Documentation for Izon qEV SMART columns: izon.com/sds

CAUTION
Sodium azide can be fatal if swallowed or in contact with skin. It can cause damage to neurological organs through prolonged or repeated exposure. It is very toxic to aquatic life with long lasting effects.

Be sure to adhere to the following guidelines and comply with any local guidelines specific to your laboratory and location regarding use and disposal.

General Precautions:

• Always wear laboratory gloves, coats, and safety glasses with side shields or goggles.
• Keep your hands away from your mouth, nose, and eyes.
• Completely protect any cut or abrasion before working with potentially infectious or hazardous material.
• Wash your hands thoroughly with soap and water after working with any potentially infectious or hazardous material before leaving the laboratory.
• Remove watches and jewellery before working at the bench.
• The use of contact lenses is not recommended due to complications that may arise during emergency eye-wash procedures.
• Before leaving the laboratory, remove protective clothing.
• Do not use a gloved hand to write, answer the telephone, turn on a light switch, or physically engage people without gloves.
• Change gloves frequently.
• Remove gloves immediately when they are visibly contaminated.
• Do not expose materials that cannot be properly decontaminated to potentially infectious or hazardous material.
• Upon completion of the tasks involving potentially infectious or hazardous materials, decontaminate the work area with an appropriate disinfectant or cleaning solution (1:10 dilution of household bleach is recommended).

Dispose of the following potentially contaminated materials in accordance with laboratory local, regional, and national regulations:

• Biological Samples
• Reagents
• Used reaction vessels or other consumables that may be contaminated

Storage
Rapid changes in temperature should be avoided, as this can introduce bubbles into the gel bed.
Unused qEV columns can be stored at room temperature. Used qEV columns can be stored at room temperature providing they have been cleaned according to the instructions in this document and stored in 20% ethanol or 0.05% w/v sodium azide. If the appropriate solutions are not available for storage at room temperature, then columns can be stored at +4 to +8 °C after use.

Disposal
Waste buffer should be disposed of in a safe manner. Sodium azide accumulation over time in copper pipes can result in an explosion.

INTRODUCTION TO SIZE EXCLUSION CHROMATOGRAPHY

Overview
Izon’s qEV SMART Size exclusion chromatography (SEC) columns separate particles based on their size as they pass through column packed with a porous, polysaccharide resin. As molecules enter the resin, smaller particles become trapped in the pores and their exit from the column is delayed (Fig 1C). As liquid exits the column, sequential fractions are collected. Particles will be distributed across the fractions based on their size, with the largest particles exiting the column first and the smallest particles exiting the column last.
The packed column is equilibrated with a buffer, which fills the column. The total column volume is occupied by both the solid resin (stationary phase) and the liquid buffer (the mobile phase). As the particles do not bind to the resin, the buffer composition will not significantly affect the resolution (the degree of separation between peaks).

Figure 1: Process of SEC (A) Schematic picture of a resin bead with an electron microscopic enlargement. (B) Schematic drawing of sample particles diffusing into the pores of the particle. (C) Graphical description of separation: (i) sample is applied to the column; (ii) the smallest particule (yellow) is more delayed than the largest particle (red); (iii) the largest particle is eluted first from the column. Band broadening causes significant dilution of the particle zones during chromatography. (D) Schematic chromatogram. From: GE Healthcare and Biosciences. (n.d.). Size Exclusion Chromatography Principles and Methods [Brochure]. Uppsala, Sweden. Accessed June 2019.

Intended Use

Izon qEV SMART columns isolate extracellular vesicles from biological samples. qEV SMART columns are equipped with RFID chips for use with the Izon Automatic Fraction Collector (AFC). These chips will
not impact manual use. The column is intended to be used in research laboratories by professional personnel for research use only. The qEV column is not intended for diagnostic purposes and should not be used to make treatment decisions.
qEV SMART columns are designed to isolate and purify vesicles from most biological samples, including:

• Serum
• Plasma
• Saliva
• Urine
• Cerebrospinal Fluid (CSF)
• Cell culture media

NOTE: most ‘raw’ samples cannot be directly run on qEV SMART columns and analysed with TRPS without some preparation such as centrifugation and concentration steps. Contact the Izon Support Centre for recommendations and protocols.

Comparison of qEV/35nm and qEV/70nm series
All qEV SMART columns are available in one of two isolation ranges, the qEV/35nm series and the qEV/70nm series. The qEV/35nm series of columns generally perform better when the target particle to be isolated is less than 110 nm in diameter, while the qEV/70nm series of columns generally perform better when the target particle to be isolated is greater than 110 nm in diameter (see Table 3). For optimal recovery of particles between 35 and 350 nm a qEV/35nm series column is recommended. For optimal recovery of particles between 70 and 1000 nm a qEV/70nm series column is recommended

Figure 2. Particle recovery by size for qEV/35 series column compared to qEV/70 series column (pink line).

Table 3: Specifications of qEV/35nm and qEV/70nm Series

 | ****

qEV/35nm SERIES

| ****

qEV/70nm SERIES

---|---|---

Target Particle Size

| ****

35 nm to 350 nm

| ****

70 nm to 1000 nm

Optimum Recovery Range

| ****

Particles < 110 nm

| ****

Particles > 110 nm

qEV2 Specifications

Table 4: qEV2 Specifications

*Izon recommends loading 2 mL to maximise purity.
**Values for human plasma samples only.

qEV2 Performance Characteristics

As shown in Figure 3 and 4 below, particles less than 70 nm typically elute later than the PCV on the qEV/70nm, whereas particles larger than 35 nm are captured in this zone on the qEV/35nm column. A higher recovery in the PCV of particles larger than 70 nm occurs on the qEV/70nm series columns compared with the qEV/35nm series (Fig. 3). Proteins typically elute slightly earlier on the qEV/35nm series. Higher protein levels in the PCV are mainly due to an increase in EV bound proteins. Figure 3: Comparison of plasma protein elution and recovery levels of 69 nm liposomes between a qEV2/35nm and a qEV2/70nm.Figure 4: Comparison of plasma protein elution and recovery levels of 200 nm particles between a qEV2/35nm and a qEV2/70nm.

qEV2 EV Elution Profile

The elution of vesicles peak at 6 mL ± 2 mL after the buffer volume, for a 2 mL sample volume and collecting 2 mL fractions. Figure 5 shows the elution of vesicles when 2 mL of plasma sample is loaded onto a qEV2/35nm column.
The majority of EVs typically elute in 8 mL. If higher purity is desired, collect only the first 6 mL. The user therefore chooses between maximising recovery by collecting a bigger volume or maximising purity by collecting a lesser volume.
The elution of plasma protein is slower, eluting predominantly from 10 – 38 mL after the buffer volume. Any vesicles recovered beyond 8 mL contain higher protein contamination and may be less suitable for downstream analysis because of their lower purity.
Indicative protein elution profiles can be obtained by monitoring the absorbance at a wavelength of 280 nm. An accurate measurement of the level of protein can be obtained using a colorimetric protein assay. Figure 5: Typical elution profile for a qEV2/35nm column with 2 mL of plasma loaded; proteins elute in later volume than vesicles. The vesicle concentration was measured using TRPS and relative protein levels by absorbance at 280 nm.

qEV2 Sample Input Volume Effects and

Recovery Rates
Effect of Sample Input Volume on EV Elution Profile
Loading higher sample volumes results in a lower level of purity in the later vesicle volumes, greater overlap between protein and EV elution peaks, and a higher protein peak within the PCV. Figure 6 shows the effect of loading increasing volumes. A sample volume of 4 mL results in some particles that elute later than the typical 8 mL PCV. Note the delay in elution of EVs in the 4 mL sample.
The optimal recommended sample volume for purity on the qEV2 is 2 mL, which consistently results in vesicles eluting in the 8 mL PCV.
Loss of vesicles occurs with sample volumes over 4 mL as the vesicle elution is broadened. EVs collected outside of the 8 mL PCV are not recommended for downstream analysis where high purity is required. Figure 6: The effect of loading larger volumes is shown in the elution profiles from loading 2 mL and 4 mL of CPC200 particles (OD600) and plasma (OD280).

Effect of Resin Type on Protein Elution Profile
The PCV on a qEV2 contains very little protein, with protein levels increasing in later volumes, as shown in Figure 7. Higher protein levels for the qEV2/35nm are due in part to higher recovery of smaller EVs in the PCV (refer to Fig. 2).

Effect of Sample Input Volume on EV Dilution and Recovery Rate
The recovery rate of input particles is dependent on the target particle size and the qEV SMART column series used. Refer to the comparison between qEV/35nm and qEV/70nm series above, Figures 2-4. The dilution factor depends on the sample loading volume and total pooled PCV. Figure 7: Percentages of protein cf. starting quantity of protein from loading 2 mL of plasma onto a qEV2. Pink bars are the PCV bars. qEV2/70nm bars are dark pink and dark blue. qEV2/35nm bars are light pink and light blue.

MANUAL OPERATING INSTRUCTIONS

The following section provides instructions for the manual use of qEV SMART columns. For use of qEV SMART columns with the Automatic Fraction Collector (AFC) instrument, please see the full AFC User Manual at support.izon.com

Operational Recommendations

The following recommendations are provided to ensure optimal performance of the qEV SMART column:

• Centrifuge samples prior to loading onto the column. To avoid clogging of column filters, it is recommended to filter or centrifuge the biological sample to remove large particulate matter.
  • Centrifuge samples at 1,500 x g for 10 minutes to remove any cells and large particles.
  • Gently move the supernatant to a new tube and centrifuge again at 10,000 x g for 10 min.
  • For microvesicle isolation, use lower g-forces for the second centrifugation step.
• Samples can be concentrated before application to the column or after isolation if needed. It is possible to concentrate samples both before and/or after use of the qEV SMART column, however Izon offers multiple column sizes to reduce the need for pre-analytical sample concentration. If concentration protocols are needed, please consider the following recommendations:
  • Concentration of some sample types may result in the formation of precipitates and protein aggregates, especially for urine samples. Concentrated samples should be centrifuged at 10,000 x g for 10 minutes prior to loading onto a qEV SMART column.
  • Izon recommends using Merck Millipore concentration devices (Amicon® Ultra Centrifugal filters; C7715). Use according to manufacturer’s recommendations.
  • Concentration of samples after purification with qEV may result in the loss of some EVs on the membrane.
• Single-use columns are advisable where the vesicles will be analysed for nucleic acids. Use of a single-use column, such as the Izon qEVsingle, will reduce the possibility of cross-contamination.
• Ensure that the sample buffer has been prepared appropriately. To maintain the functionality of EVs, the flushing buffer should be of the same temperature as the sample buffer. SEC can also be used to exchange the buffer of a sample.
  • Sample buffer temperature should be within the operational temperature of 18-24 ˚C (65-75 ˚F).
  • Sample buffers should be degassed and room temperature to avoid air bubbles forming in the gel bed. Rapid changes in temperature, for example removing packed columns from a cold room and applying buffer at room temperature, can introduce air bubbles in the packed bed, resulting in poorer separation.
  • Use a buffer with an ionic strength of 0.15 M or greater to avoid any unwanted ionic interactions between the solute molecule and the matrix.
  • Only use freshly filtered (0.22 µm) buffer to avoid introducing particulate contamination.
  • qEV SMART columns come equilibrated in filtered PBS containing < 0.1% w/v sodium azide.

Column Setup and Equilibration

1. Equilibrate the column and the sample buffer to be within the operational temperature range of 18-24 ˚C.
   CAUTION
   Do not remove the column caps until the column has reached operational temperature.
   Sample buffers should be degassed and room temperature to avoid air bubbles forming in the gel bed.

2. Attach the column in an upright position to a stand ready for use. The Automatic Fraction Collector (AFC) is available from store.izon.com

3. Rinse the reservoir with buffer.

4. Before connecting the reservoir to the column, add 5 mL of buffer to the reservoir and wait for the frit to wet and buffer to start running through.

   • If frit is slow to wet, apply some pressure to the reservoir top with the palm of your hand to aid the flow.

5. Allow buffer to run until it stops at the frit.

6. Remove the column connector cap, top up the connector with buffer, and firmly attach (a good seal is critical) the loading reservoir to the connector being careful to avoid trapping air bubbles in the connector.

7. Add buffer to the reservoir.

8. Remove the bottom cap and allow the buffer to start running through the column.

9. Flush the column with at least one column volume of sample buffer. If an elution buffer other an PBS is to be used, equilibrate the column with at least 3 column volumes of the new buffer.
   Only use freshly filtered (0.22 µm) buffer to avoid introducing particulate contamination.

Sample Loading

1. To avoid clogging of column filters, it is recommended to filter or centrifuge the biological sample to remove large particulate matter. See Section 4.1: Operational Recommendations for more information.

2. Continue to allow buffer to run through the column. The column will stop flowing when all of the buffer has entered the loading frit.

3. Load the prepared centrifuged sample volume onto the loading frit.
   CAUTION
   Avoid stopping the column flow during the run for long periods of time to ensure accurate EV separation.

4. Immediately start collecting the buffer volume (this includes the volume displaced by loading the sample).

5. Allow the sample to run into the column. The column will stop flowing when all of the sample has entered the loading frit.

6. Top up the column with buffer and continue to collect the buffer volume.
   NOTE
   To collect accurate volumes, only load the required volume to the top of the column, wait for the volume to run through until the flow stops and repeat.
   Different samples may give slightly different elution profiles and purity, hence an initial measurement of EV concentration and protein contaminants in collected fractions is recommended.

Column Flush and Storage

1. After all the desired fractions have been collected follow the cleaning protocol outlined subsequently, before loading another sample.
2. If storing the column for future use, perform the cleaning procedure with buffer containing a bacteriostatic agent (e.g. 0.05 % w/v sodium azide or 20% ethanol).
3. Columns can be stored at room temperature after use, providing
   they have been cleaned according to the instructions above. If the appropriate solutions are not available then columns can be stored at + 4 to +8 ˚C after use.

Restoring Column Flow After Blockage due to Airlock in the Junction

1. Place the bottom cap on the column.
2. Remove the loading reservoir.
3. Unscrew the column cap and add buffer to the top frit until the buffer is level with the top edge of the column.
4. Screw the column cap back on forcing buffer up through the connector junction.
5. Add 2 mL of buffer to the loading reservoir and allow buffer to run through until it stops at the frit.
6. Carefully attach the loading reservoir to the connector being careful to avoid trapping any air bubbles in the connector.
7. Add more buffer to the loading reservoir before removing the bottom cap.
8. The column should begin to flow again.

RESOURCES

Column Cleaning and Sanitisation
Sanitise and remove precipitated proteins, non-specifically bound proteins and lipoproteins by rinsing the column with 100 mL of buffer, washing the column with 2 mL of 0.5 M NaOH, then flush with at least 100 mL of buffer until the pH of the eluted buffer is the same as the starting wash buffer pH. The pH will return to neutral after about 2-3 column volumes of wash buffer.

Protocols for EV Isolation from Common Sources
See Izon Support Centre support.izon.com for application notes and typical protocols for common EV samples. If you are unsure of what to do to prepare your sample, please contact support@izon.com for assistance.

EV Analysis Using TRPS
Izon recommends TRPS analysis for determination of particle size, concentration, and zeta potential. The Izon Reagent Kit includes coating solutions for pre-coating the pore, minimising non-specific binding and provides for stable and accurate sizing and concentration analysis.
For TRPS analysis of the EVs, Izon recommends an initial dilution of 1/5 or 1/10 in electrolyte. Optimise the dilution to achieve a rate at the highest operating pressure of approximately 200 to 1600 particles per minute to avoid pore blockage.
See Izon Support Centre support.izon.com for more information on the analysis of EVs with TRPS.

www.izon.com

References

• Izon Science Safety Data Sheets (SDS)
• Izon Science Store
• Izon Science Support Centre
• Isolate & Measure Extracellular Vesicles & Nanoparticles

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