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To suppress adsorption of extracellular vesicles to laboratory tools

EV-Save™ Extravellular Vesicle Blocking Reagent

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EV-Save™ Extracellular Vesicle Blocking Reagent protects extracellular vesicles (EVs) against freezing and suppresses their adsorption to labware such as test tubes and pipette tips. Our product lineup also includes EV-Save™ for in vivo (Code No. 050-09461) use, which consists only of ingredients that have been successfully used as pharmaceutical additives.

Precautions for use
    • EV-Save™ series are not effective in serum, plasma, or samples containing a lot of impurities.
    • EV-Save™ (Code No. 058-09261) contains a polymer. Do not use EV-Save™ if the polymer may affect the experimental results of subsequent processes.
    • Using EV-Save™ in conjunction with fluorescent dye could raise the background level.
    • EV-Save™ does not affect the following analyses using EVs:
      1. Nanoparticle tracking analysis (NTA)
      2. ELISA
      3. Experiments involving the addition of EVs to cells
      4. Western blotting*1
      5. Microarray analysis*1

        *1 Applicability confirmed for EV-Save™ (Code No. 058-09261) only.

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Procedure

Using MagCapture™ Exosome Isolation Kit PS Ver.2 (code No. 290-84103)

For isolation of extracellular vesicles from cell culture supernatants

  1. Add EV-Save™ or EV-Save™ for in vivo to cell culture supernatants at a final dilution of 1:100 and mix by inverting or tapping. If used for concentrating cell culture supernatants, add EV-Save™ to cell culture supernatants before ultrafiltration (EV-Save™ for in vivo is not suitable ultrafiltration).
  2. Proceed with the experiments to Step 5. "Washing of EVs-binding beads" described in the MagCapture™ Exosome Isolation Kit PS Ver.2 (Ver.2) instructions.
  3. Add EV-Save™ or EV-Save™ for in vivo to "Exosome Elution Buffer" provided with Ver.2 at a final concentration of 1:100 and mix by inverting or tapping.
  4. Restart the experiment at Step 6. "Elution of Extracellular Vesicles" described in the Ver. 2 instructions, to elute extracellular vesicles.
  5. Store the eluted extracellular vesicles*2 or proceed to the next experiment.

For isolation of extracellular vesicles from serum and plasma

  1. Proceed to Step 5. "Washing of EVs-binding beads" described in the Ver. 2 instructions.
  2. Add EV-Save™ or EV-Save™ for in vivo to "Exosome Elution Buffer" provided with Ver.2 at a final concentration of 1:100 and mix by inverting or tapping.
  3. Restart the experiment at Step 6. "Elution of Extracellular Vesicles" described in the Ver. 2 instructions, to elute extracellular vesicles.
  4. Store the eluted extracellular vesicles*2 or proceed to the next experiment.

Using another method of isolation

For isolation of extracellular vesicles from the cell culture supernatants

  1. Add EV-Save™ or EV-Save™ for in vivo to cell culture supernatants at a final dilution of 1:100 and mix by inverting or tapping.
  2. Add EV-Save™ or EV-Save™ for in vivo isolated extracellular vesicles at a final concentration of 1:100 and mix by inverting or tapping.
  3. Store the eluted extracellular vesicles*2 or proceed to the next experiment.

*2: By adding this product, the sample can be stored frozen at under −20°C. However, please avoid a large number of freeze-thaw cycles.

EV-Save™ Extracellular Vesicle Blocking Reagent

Data 1: Anti-adsorption effect of EV-Save™ -tubes-

Using the PS affinity method*3, exosomes were isolated from COLO201 cell conditioned medium. A suspension of exosomes at 3 ng/μL was placed in a tube, allowed to stand for 3 minutes, and then 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 the PS Capture™ Exosome ELISA Kit (Anti Mouse IgG POD) (Code No. 297-79201), CD63 signal was measured to determine the reduction rate of exosomes along with the number of sample transfers. The results were reflected in a graph setting the initial CD63 signal as 100%.

*3: EVs are captured by phosphatidylserine(PS)-binding proteins and metal ions, and eluted with a chelating reagent.

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Loss of exosomes associated with transfers was observed with either conventional tubes or low-protein-adsorption tubes.
Addition of EV-Save™ almost completely suppressed exosome loss.

Data 2: Anti-adsorption effect of EV-Save™ -Combination with PS affinity method-

Study of EV-Save™ effects on loss of exosomes in the exosome purification process from TIG3 cell conditioned medium using the PS affinity method.

experimental conditions

Using the PS affinity method, exosomes were purified from 1 mL of conditioned medium from COLO201, TIG3, and human iPS cell cultures. 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 the kit with and without EV-Save™ added".

Using the PS Capture™ Exosome ELISA Kit (Anti Mouse IgG POD) (Code No. 297-79201), CD63 signal was 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%.

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− − 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™

Exosomes are damaged by repeated freeze-thaw cycles (Witwer, K. W. et al., 2013). We examined whether such damage to exosomes can be suppressed by the addition of EV-Save™.

Experimental conditions

COLO201 cell-derived exosomes purified by PS affinity method were freeze-thawed, prior to be analyzed with the PS Capture™ Exosome ELISA Kit (Anti Mouse IgG POD) (Code No. 297-79201) (A) and by transmission electron microscopy (TEM) (B).

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

Data 4: Prevention of extracellular vesicle loss during ultrafiltration

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

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Extracellular vesicles are not lost during ultrafiltration.

Data 5: Use in cell culture

EV-Save™ was added to a TBS solution containing human iPS cells at a ratio of 1:100. The resultant TBS solution was then added to culture medium at a ratio of 1:5. Human iPS cells 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%).

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No effect on proliferation of human iPS cells.

EV-Save™ Extracellular Vesicle Blocking Reagent for in vivo

Comparing EV-Save™ and EV-Save™ for in vivo

EV-Save™ (Code No. 058-09261) EV-Save™ for in vivo (Code No. 050-09461)
Excretion to outside the body No data Only ingredients with molecular weights that allow the product to be excreted outside the body
Published use as pharmaceutical additives No Yes
Use in animal experiments No data Possible
Ultrafiltration application Possible Impossible

Application Data

Data 1: Protective effect against exosome freezing

EV-Save™ and EV-Save™ for in vivo were each added to COLO201-cell-derived exosomes, previously purified using a PS affinity method, and freeze-thawing was performed either once or for three repeated cycles. Thereafter, the exosome content in the sample tube was measured using the PS Capture™ Exosome ELISA Kit (Streptavidin HRP) (code No. 298-80601).

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Without addition of EV-Save™, the exosome marker CD9 signal was decreased by freeze-thawing, and this signal reduction was suppressed by adding EV-Save™ and EV-Save™ for in vivo.

Data 2: Suppressive effect against exosome adsorption

EV-Save™ and EV-Save™ for in vivo were each added to COLO201-cell-derived exosomes, previously purified using a PS affinity method, and the sample was stored at 4°C for 16 hours. Thereafter, the exosome content in the sample tube was measured using the PS Capture™ Exosome ELISA Kit (Streptavidin HRP).

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Without addition of EV-Save™, the exosome content (CD9 signal) decreased, and this was suppressed by adding EV-Save™ and EV-Save™ for in vivo.

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