EV-Save(TM) Extracellular Vesicle Blocking Reagent
- for Genetic Research
- Manufacturer :
- FUJIFILM Wako Pure Chemical Corporation
- Storage Condition :
- Keep at -20 degrees C.
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1mL
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In stock in Japan |
Document
Product Overview
EV-Save™ Extracellular Vesicle Blocking Reagent is a polymeric reagent that prevents EV from adsorbing to tubes, pipette tips, and other labware. It prevents loss of EVs due to adsorption during processing and storage, thereby increasing the yield. Addition of this product is highly recommended prior to ultrafiltration, purification, and storage.
Features
- Prevents adsorption of EV in cell culture supernatants and purified EV onto laboratory equipment
- Protects EV from freeze-thaw damage
- Easy to operate, just add to samples
Sample (Recommended)
- Cell culture supernatant
- Purified EV
[Note]
The anti-adsorption effect may be reduced in serum, plasma an samples containing high levels of interfering substances.
Impacts on Analysis
No interference with the analysis of EVs in the following analyses:
- - Nanoparticle Tracking Analysis (NTA)
- - Western blotting
- - ELISA
- - Microarray
- - Cell culture
[Note]
- This product contains polymers. Do not use this product if the presence of polymers may affect experimental results in subsequent processes
- When administering EVs to experimental animals, please use EV-Save™ Extracellular Vesicle Blocking Reagent (Product No. 050-09461), which is designed specifically for in vivo applications.
Protocol
Before EV Isolation
Add this product to the cell culture supernatant at a 1:100 dilution and mix gently by inversion or tapping. (If this cell culture supernatant is to be concentrated by ultrafiltration, add this product at a 1:100 dilution to the cell culture supernatant prior to concentration.)
After EV Isolation
Add this product to the isolated EV solution at a 1:100 dilution and mix gently by inversion or tapping. When isolating EVs using the MagCapture™ Exosome Isolation Kit PS Ver.2, add this product to Exosome Elution Buffer supplied with the kit at a 1:100 dilution and mix gently by inversion or tapping.
For details, please refer to the package insert.
Data
Anti-adsorption Effect
Inhibition of EV Adsorption during Tube Transfer
EV-Save™ was added to COLO201 cell-derived EVs purified by the PS affinity method and allowed to stand for 3 minutes. The sample was then transferred to another tube, and the transfer was repeated. The amounts of EV (CD63 signal) in the sample tubes were measured by PS Capture™ Exosome ELISA Kit (Anti Mouse IgG POD).
[Result]
CD63 signal decreased with transfer. The addition of EV-Save™ almost completely prevented the decrease in signal caused by the transfer.
Inhibition of EV Adsorption with Low Protein-Binding Tubes
EVs derived from COLO201 cells, purified by the PS affinity method, were diluted to 3 ng/μL and placed in low protein-binding tubes for 3 minutes. The samples were then transferred to new tubes, and this process was repeated four times. EV content (CD63 signal) in each sample tube was measured using the PS Capture™ Exosome ELISA Kit (Anti-Mouse IgG POD).
[Result]
Using low protein-binding tubes alone had little or no effect in inhibiting EV adsorption in this experiment. In contrast, the addition of EV-Save™ effectively suppressed the reduction in CD63 signal caused by repeated tube transfers in both standard tubes and low protein-binding tubes.
Inhibition of EV Adsorption During Ultrafiltration
EVs purified from bone marrow-derived MSC culture supernatant using the PS affinity method were subjected to ultrafiltration using Vivaspin (100 kDa). The ability of EV-Save™ to reduce EV loss during this process was evaluated.
[Result]
The addition of EV-Save™ suppressed the reduction in CD63 signal caused by ultrafiltration.
Combination with the PS Affinity Method
A study was conducted to determine whether the addition of EV-Save™ could prevent loss of EV during the purification from TIG-3 cell culture supernatants using the PS affinity method.
[Result]
When EV-Save™ was added to the cell culture supernatant and the elution buffer of the PS affinity method (+/+), EV yield was increased by about 20% compared to without the addition of EV-Save™ (－/－).
Cryoprotection Effect
EV-Save™ was added to COLO201 cell-derived EVs purified by the PS affinity method and freeze-thawed. The amounts of EV (CD63 signal) in the sample tubes were measured by PS Capture™ Exosome ELISA Kit (Anti Mouse IgG POD), and their structure was analyzed by transmission electron microscopy (TEM).
(1) ELISA
(2) Transmission electron microscopy
[Result]
The CD63 signal decreased with freeze-thawing. The addition of EV-Save™ prevented the decrease in signal caused by freeze-thawing. Observation by electron microscopy confirmed that the reduction in particle numbers was suppressed.
Effect on Cell Viability
A TBS solution containing this product at a 1:100 dilution was added to culture medium at a ratio of 1:5. Human iPS cells were cultured in this medium for 3 days, and cell viability was determined.
[Result]
The addition of EV-Save™ had little or no effect on the viability of human iPS cells.
FAQ
About sample
- Does the performance of this product vary depending on the composition or pH of the sample?
- We have not confirmed how specific components or the pH of a sample affect performance. However, in samples containing serum, plasma, or high levels of contaminants, the components in the sample may prevent the adsorption of EVs, making it difficult to achieve the anti-adsorption effect intended by adding this product.
About application
- How long can EVs with this product added be stored?
- We have confirmed that when this product is added to EV isolated using the PS affinity method, they can be stored at -20°C for at least 12 months.
(Please note that the storage period may vary depending on the sample and storage conditions, so this should be considered a general guideline only.)
- Is it possible to use purified EV samples for proteome analysis after this reagent has been added?
- It is possible after EV-Save™ is removed by the following method:
- Add 5 times the volume of cold acetone to purified EVs and mix by inversion.
- Allow to stand at -20°C for 1 hour.
- Centrifuge at 1,500 × g for 10 min at 4°C.
- Remove the supernatant.
- Repeat steps 1 - 4 three times.
- Open the lid and allow to air dry for 1 hour (RT).
- Dissolve in 20 μL of 9 M urea, 20 mM HEPES-NaOH, pH 8.2.
- When quantifying total protein in purified EV samples containing this product, which method should I choose?
- We recommend using the BCA method (Bicinchoninic Acid Assay) to quantify total protein in purified EV samples to which this product has been added.
- Does adding this product affect the staining of EVs using fluorescent dyes?
- Although this product does not contain fluorescent substances, adding it may increase background levels during staining with fluorescent dyes. To accurately determine whether signal is present, we recommend comparing each sample with an appropriate control.
- Can this product be used to inhibit adsorption in tangential flow filtration (TFF)?
- We have not verified the use of this product in TFF. If you wish to prevent EV adsorption to membranes or other surfaces during TFF, please use the EV-Save™ Extracellular Vesicle Blocking Reagent for TFF (Product No. 059-09671).
Overview / Applications
| Outline | This product is for research use only. Do not administer it to human.It is a polymeric reagent that suppresses adsorption of extracellular vesicles such as Exosome in cell culture supernatant to laboratory instruments/containers. Since extracellular vesicles adsorb to sample tubes and ultrafiltration membranes, they are significantly reduced during purification and storage. This product strongly suppresses adsorption of such extracellular vesicles. When MagCapture (TM) Exosome Isolation Kit PS (Code No. 299-77603, 293-77601)is used to purify extracellular vesicles from cell culture supernatant, use of this product is strongly recommended. [Before use] ·If you use this product for serum, plasma or contaminated samples, the effect of this product will not be obtained. ·This product contains polymer. Do not use this product if there is a risk that the existence of polymer will affect the results of the experiment in the subsequent process. |
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Property
| Appearance | Colorless - nearly colorless, clear liquid |
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| Density | 1.005 - 1.011g/mL (20 degrees C) |
Manufacturer Information
Alias
- EV-Save (TM) Exosome Blocking Reagent
For research use or further manufacturing use only. Not for use in diagnostic procedures.
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