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To suppress adsorption of extracellular vesicles to laboratory tools

EV-Save™ Extracellular Vesicle Blocking Reagent

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EV-Save™ Extracellular Vesicle Blocking Reagent is a polymer reagent to prevent adsorbing extracellular vesicles in cell culture supernatants to laboratory tools such as tubes and pipet tips, which reduces the loss of extracellular vesicles during experiments and storage. Add into samples before ultrafiltration of cell culture supernatants, isolation and storage of extracellular vesicles.

Precautions for use
  • The effect of EV-Save™ cannot be obtained when it is used for serum, plasma, or samples containing a lot of impurities.
  • The product contains a polymer. Do not use EV-Save™ if the polymer may affect the experimental results in the post process.
    It have been confirmed that there are no effect by EV-Save™ in the following analyses using extracellular vesicles.
    1. Nanoparticle Tracking Analysis
    2. Western blot
    3. ELISA
    4. Microarray analysis
    5. Cell culture
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Features

  • Strong suppression of adsorption of extracellular vesicles in culture supernatant and after purification to laboratory tools
  • Simple operation just to add to the sample

Data 1

Anti-adsorption effect of EV-Save™ ― Tubes ―

Whether EV-Save™ could suppress adsorption of purified exosomes to tubes or not was investigated.

Experiment conditions

Using MagCapture™ Exosome Isolation Kit PS (Code No. 293-77601), exosomes were isolated from COLO201 cell conditioned medium. A suspension of exosomes at 3 ng/μL was placed in a tube and allowed to stand for 3 minutes. Then, the suspension was transferred to another tube. Such standing-transfer operation was repeated 4 times. In the “With EV-Save™” condition, EV-Save™ was initially added to the suspension to achieve 100-fold dilution in volume before the standing-transfer operations.

Using PS Capture™ Exosome ELISA Kit (Anti Mouse IgG POD) (Code No. 297-79201), CD63 signals of the exosomes were measured to determine the reduction rate of exosomes with the number of transfers. The results were reflected in a graph setting the initial CD63 signal as 100% relatively.

Loss of exosomes occurred associated with transfers using either general tubes or low-protein-adsorption tubes. Addition of EV-Save™ almost completely suppressed loss of exosomes associated with tube transfers.

Data 2

Anti-adsorption effect of EV-Save™ ― Combination with MagCapture™ Exosome Isolation Kit PS ―

Whether EV-Save™ would suppress loss of exosomes in an exosome purification process from TIG3 cell conditioned medium using MagCapture™ Exosome Isolation Kit PS or not was investigated.

Experiment conditions

Using MagCapture™ Exosome Isolation Kit PS, exosomes were purified from 1 mL of the conditioned medium each of COLO201, TIG3, and human iPS cells. The conditioned medium was not subjected to concentration by ultrafiltration. Exosomes were purified from the“ conditioned medium with and without EV-Save™ added” using “‘Exosome Elution Buffer’ included in MagCapture™ Exosome Isolation Kit PS with and without EV-Save™ added.”

Using PS Capture™ Exosome ELISA Kit (Anti Mouse IgG POD) (Code No. 297-79201), CD63 signals of exosomes were measured as an indicator of the exosome amount. The results were reflected in a graph setting the CD63 signal in the conditioned medium as 100% relatively.



− − Without EV-Save™ added to conditioned medium/ without EV-Save™ added to“ Exosome Elution Buffer”
− + Without EV-Save™ added to conditioned medium/ with EV-Save™ added to“ Exosome Elution Buffer”
+ + With EV-Save™ added to conditioned medium/ with EV-Save™ added to“ Exosome Elution Buffer”

The yield of exosomes from the process with EV-Save™ added to both TIG3 cell conditioned medium and “Exosome Elution Buffer” included in MagCapture™ Exosome Isolation Kit PS (+/+) was 20% higher than that from the process without EV-Save™ added to either (−/−).

Data 3

Cryoprotective effect of EV- Save™

It is known that exosomes are damaged by repeated freeze-thaw (Witwer, K. W. et al., 2013). It was examined whether such damage for exosomes could be suppressed by the addition of EV-Save™.

Experimental conditions

The COLO201 cell-derived exosomes purified by MagCapture™ Exosome Isolation Kit PS were freezethawed. Thereafter, measurement with a PS Capture™ Exosome ELISA Kit (Anti Mouse IgG POD) (Code No. 297-79201) (A) and analysis with a transmission electron microscope (TEM) were performed (B).

Although freeze-thaw reduced CD63 signal, such reduction was suppressed by the addition of EV-Save™ (A). Furthermore, from the result of electron microscopic analysis (B), it was confirmed that freezing and thawing caused a marked decrease in the number of particles, but the suppression effect by EV-Save™ was also recognized for such a phenomenon.

Data 4

Decrease in extracellular vesicles during ultrafiltration is prevented

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Reduction in extracellular vesicles after ultrafiltration for concentration (Vivaspin20, 100K dalton) of human iPScell culture supernatant was evaluated using PS Capture™ Exosome ELISA Kit (Anti Mouse IgG POD). Graph shows CD63 signal relative to that obtained before ultrafiltration (100%).





Extracellular vesicles are not lost during ultrafiltration

Data 5

Use in cell experiments

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To TBS solution containing human iPScells, EV-Save™ was added at a ratio of 1/100, and the resultant TBS solution was added to culture medium at a ratio of 1/5. Human iPScells were cultured on this medium for 3 days, and viable cell count was determined. Graph shows CD63 signal relative to that obtained from a sample without EV-Save™(100%).



No effect on proliferation or pluripotency of human iPS cells

Procedure

For isolation of extracellular vesicles from cell culture supernatants using Mag Capture™ Exosome Isolation Kit PS (Code No. 299-77603, 293-77601)

  1. Add EV-Save™ Extracellular Vesicle Blocking Reagent to be diluted 100- fold into cell culture supernatants and mix it by inverting or tapping.
    In the case of concentrating cell culture supernatants by an ultrafiltration unit, add EV-Save™ Extracellular Vesicle Blocking Reagent into cell culture supernatants before ultrafiltration.
  2. Proceed with an experiment to the step of “Elution of extracellular vesicles” described in the instruction of MagCapture™ Exosome Isolation Kit PS.
  3. Add EV-Save™ Extracellular Vesicle Blocking Reagent to be diluted 100- fold into “Exosome Elution Buffer” accompanied with Mag Capture™ Exosome Isolation Kit PS and mix it by inverting or tapping.
  4. Elute extracellular vesicles according to the instruction of Mag Capture™ Exosome Isolation Kit PS.
  5. Store the eluted extracellular vesicles, or proceed to the next experiment.

For isolation of extracellular vesicles from cell culture supernatants using other methods

  1. Add EV-Save™ Extracellular Vesicle Blocking Reagent to be diluted 100- fold into cell culture supernatants and mix it by inverting or tapping.
  2. Add EV-Save™ Extracellular Vesicle Blocking Reagent to be diluted 100-fold into isolated extracellular vesicles and mix it by inverting or tapping.
  3. Store it or proceed to the next experiment

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