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Exosome Related Products Q&A


MagCapture™ Exosome Isolation Kit PS

PS Capture™ Exosome ELISA Kit (Anti Mouse IgG POD)


MagCapture™ Exosome Isolation Kit PS

Kit specifications and performance

Q1: Could you tell me the principle of this purification method?

This kit for purification of extracellular vesicles (EVs) including exosomes utilizes a protein called Tim4 that binds phosphatidylserine (PS), a phospholipid located on the surface of EVs including exosomes, in a manner dependent on metal ion.
This is an affinity purification technique not using an antibody.



Q2: What kind of EVs are purified using this kit?

Exosomes and microvesicles with PS exposed on the surface of lipid membrane are purified using this kit.



Q3: What is the difference between exosomes and microvesicles?

Exosomes and microvesicles are distinguished from each other by difference in generation pathway.
Exosomes are defined as extracellular vesicles secreted from late endosomes, while microvesicles as extracellular vesicles directly budding from cell membrane.
Exosomes and microvesicles differ in size distribution: exosomes are considered to have a particle size of approximately 40-100 nm, while microvesicles have a particle size of approximately 100-1,000 nm although much smaller microvesicles have been reported. Thus, exosomes and microvesicles may not be clearly distinguished by size.



Q4: Can exosomes and microvesicles be purified separately?

As described above, exosomes and microvesicles cannot be clearly distinguished by size and their complete separation by size is impossible. We recommend the following method for preliminary separation of exosomes and microvesicles to obtain major fractions of individual vesicles using this kit. To purify extracellular vesicles with a smaller particle size (small EVs) including exosomes, use the supernatant obtained after centrifugation at 10,000 × g as sample.
To purify extracellular vesicles with a larger particle size (large EVs) including microvesicles, collect the supernatant obtained after centrifugation at 1,200 × g, centrifuge this supernatant at 10,000 × g to isolate the precipitate, suspend the precipitate with TBS, and use this suspension as sample.
To purify both vesicles together, use the supernatant obtained after centrifugation at 1,200 × g as sample.
See the instruction manual for details of sample pre-treatment conditions.



Q5: Is there anything else co-purified with exosomes and microvesicles?

Enveloped viruses, if present in sample, are known to be recovered together with exosomes and microvesicles. This is because enveloped viruses have PS exposed on the viral membrane surface. Utilizing this property, this kit may potentially be applied to recovery of enveloped viruses.
Separation of enveloped viruses from exosomes and microvesicles recovered using this kit requires affinity purification using a virus-specific antibody.
This applies also to purification methods using antibodies against exosome markers located on the envelope (e.g., CD63).



Q6: Do all exosomes have PS exposed on the membrane surface?

No finding that all exosomes have PS exposed on the membrane surface has been obtained to date. Although PS has been identified on exosomes from many cells (e.g., mouse oligodendrocytes) used in articles analyzing membrane lipid components of exosomes, possible existence of exosomes with little or no PS exposed on the surface cannot be ruled out. This kit cannot recover such exosomes.
The data posted on the website <Reference data: Qualitative analysis of EVs purified from various cell culture supernatants> presents results of detection of exosome markers (CD9, CD63, CD81) per ng of exosomes purified using this kit. Although the signal intensities vary due to difference in expression levels of exosome markers depending on cell species, the figure shows that exosomes in cell culture supernatants were successfully purified from 13 cells using this kit.
Although no finding that all exosomes have PS exposed on the membrane surface has been obtained, the PS affinity method used in this kit is considered superior in terms of exosome capture/purification compared with using antibodies against exosome markers, because the expression level of individual exosome markers greatly varies depending on cell species.



Q7: From what samples are exosomes purified using this kit?

We have experiences of successful exosome recovery from cell culture supernatant, serum, heparinized plasma, EDTA-plasma, and urine. However, since this method requires metal ion for affinity reaction of exosomes, exosome recovery from plasma treated with a chelating agent (EDTA) requires buffer exchange in advance by ultrafiltration etc.
Please visit our website for detailed protocol. User-reported applications of this kit include exosome purification from spinal fluid and saliva.



Q8: How much is the number of exosomes recovered per purification?

Although it greatly varies depending on the type and amount of sample, we experienced recovery of approximately 30 µg/mL protein (as measured by BCA method) and 1-2 × 1010 particles/mL (as measured using NanoSight LM10) per purification (by single purification from K562 cell culture supernatant collected after enhancement of exosome secretion with monensin sodium salt and subsequently concentrated from 5 mL to 1 mL). We also experienced recovery of approximately 34 µg/mL protein (as measured by BCA method) and 5 × 109 particles/mL (as measured using NanoSight LM10) per purification from 1 mL of pooled human normal serum. This kit yields an eluate in a final volume of 100 µL.



Comparison with conventional methods

Q9: What are advantages of this kit over ultrafiltration?

This kit is capable of easier and more reproducible exosome recovery at a higher purity and efficiency than those of ultrafiltration. This kit is also confirmed to be capable of recovering exosomes difficult to precipitate by ultracentrifugation.
The purity of exosomes recovered using this kit is high: an exosome fraction at a high purity comparable to that of exosome isolated by a combination of ultrafiltration and density gradient centrifugation.



Q10: What are advantages of this kit over the affinity method using antibodies?

While the affinity method using antibodies uses antibodies against exosome surface antigens and therefore requires exosome recovery by dissociation of bound exosomes by elution with a denaturant or under an acidic condition, this kit allows elution of bound exosomes under a neutral condition with a chelating agent and recovery of almost intact exosomes. Since no denaturant is required for elution, the resulting exosome contains less amount of contaminating proteins non-specifically adsorbed on magnetic beads and is recovered at a higher purity. Another confirmed advantage of this method is a high recovery efficiency.
Compared with the conventional antibody affinity method targeting a single exosome surface marker protein, this kit targeting a membrane lipid component is expected to capture a wider range of exosomes. In addition, although antibodies recognizing surface marker proteins may fail to recognize homologous antigens from different animal species, this kit is applicable to a wide range of animal species (we have experiences of its application to human, mice, cattle, and monkeys).



Q11: What are advantages of this kit over polymer precipitation?

Compared with polymer precipitation, this kit yields exosomes at a higher purity, although at a lower recovery efficiency.



Operational procedures and composition of the kit

Q12: How long is the operation time for exosome purification using this kit?

Sample pre-treatment requires approximately 1 hours (buffer exchange of 1 mL EDTA-plasma requires approximately 3-4 hours), while the entire kit process takes 3 hours and 30 minutes including immobilization of Exosome Capture onto magnetic beads for approximately 15 minutes, incubation with sample for 3 hours, and washing plus elution of exosomes for approximately 35 minutes. Although the time for incubation with sample may be reduced to 2 hours, make sure in advance to confirm that this change does not affect experimental results.



Q13: What are the steps for operation of this kit requiring particularly careful manipulation?

(1) At the final step of washing after incubation of Exosome Capture-immobilized magnetic beads with sample, thoroughly remove the washing buffer. Do not proceed to elution until complete removal of the washing buffer is confirmed.
(2) At the elution step, after addition of the elution buffer, thoroughly suspend the beads to ensure that no beads remain aggregated.



Q14: What is the composition of Elution Buffer?

It is a Tris-based buffer solution containing 1 mM chelating agent, salt, and a preservative agent. If any of these components may interfere with subsequent analysis, change to an appropriate buffer by ultrafiltration (Sartorius VivaSpin500, molecular weight cutoff 100K, Product No.: VS0141).



Q15: Are used magnetic beads with immobilized Exosome Capture compatible with recycling?

Yes. Used magnetic beads are reusable up to 4 times after regeneration to ensure efficient recovery of exosomes in sample. The kit includes all necessary buffers in sufficient amounts, and the kit specifications allow reuse of magnetic beads up to 50 times in cases of repeated extraction from an identical sample or no concern of contamination. Reuse is recommended for recovery from a sample with a volume of 1 mL or larger or a concentrated sample. See the instruction manual for details.



Q16: Are Exosome Capture-immobilized magnetic beads compatible with storage?

Yes. If Exosome Capture-immobilized magnetic beads after elution of exosomes are to be reused, store them refrigerated in Washing Buffer included in the kit or TBS (Wako experience: usable after storage for 3 months).



Q17: Is there any step that can be carried over to next day?

Incubation of the Exosome Capture-immobilized magnetic beads with sample (for 3 hours in the standard protocol) may be prolonged up to overnight without any problem.



Sample volume

Q18: How much is the minimum sample volume required for a single purification?

To assure consistent mixing of magnetic beads and the sample solution, the sample volume should be 500 µL or larger for mixing with a rotator and 100 µL or larger for mixing with a tube mixer. When the sample volume is smaller than the relevant lower limit, add TBS to make a sample volume exceeding it before incubation with the Exosome Capture-immobilized magnetic beads.



Q19: Is this kit compatible with recovery from large-volume samples?

Yes, this kit is compatible with large-volume samples after concentration. For cell culture supernatant, it is compatible with a sample volume up to 50 mL. Concentrate 50 mL of supernatant pre-treated by centrifugation to 1 mL by ultrafiltration (filter recommended: Sartorius VivaSpin20, molecular weight cutoff 100K, Product No.: VS2041).
It is compatible not only with serum-free medium but also with 10% FBS-supplemented medium. See the instruction manual for details.
Since serum samples cannot be concentrated, this kit is compatible with a serum sample volume only up to 10 mL. However, due to reduction in recovery efficiency at a serum sample volume larger than 1 mL, repeated purification with used magnetic beads are recommended in such cases.



Analysis after exosome extraction

Q20: What are the components of exosomes?

Exosomes are reported to contain proteins, lipids, and nucleic acids (DNA, microRNA, mRNA) and others.



Q21: For what kind of analyses can I use the extracellular vesicles purified using this kit?

Since intact extracellular vesicles are obtained using this kit, you can use them for any analysis.

(Examples)
• Protein analysis: protein electrophoresis, Western Blotting, mass spectrometry, flow cytometry, ELISA, etc.
• Nucleic acid analysis: qPCR, microarray, next-generation sequencing, etc.
• Particle analysis: electron microscopy, NanoSight (NTA), etc.
• Functional analysis: in vitro/in vivo administration experiments, etc.



Q22: Can I use the extracellular vesicles purified with this kit for uptake assay to cells without any pre-treatment?

After preparing TBS buffer containing 2 mM EDTA, please use instead of the elution buffer supplied with the kit. For the exosome sample obtained in the elution step, sterilization treatment using a centrifugal filter unit (Millipore Ultrafree - MC, GV 0.22 µm, sterile, Catalog No.: UFC30GV0S) is recommended. After sterilization treatment, use it for uptake assay etc.



Q23: How much is the exosome amount required for electron microscopic analysis?

We have an experience of electron microscopic analysis using 2-4×1010 exosome particles (measured using NanoSight LM10).



Q24: How can I store the extracellular vesicles purified with this kit?

Store refrigerated or frozen. Store at − 80 °C if long-term storage is desired. For freeze preservation, storage in aliquots is recommended to avoid freezingthawing.



Q25: How can I perform Western Blotting analysis using the exosome extract recovered?

At our Laboratories, 15 µL of eluate and 5 µL of 4 × SDS sample buffer are mixed and applied for SDS-PAGE.



Exosome markers

Q26: How are the exosomes purified using this kit identified?

They have been identified by Western Blotting using antibodies against exosome surface antigens, electron microscopy, density gradient centrifugation, and particle size measurement (NanoSight LM10), etc.



Q27: What are the marker proteins identified in the purified exosomes by Western Blotting?

They include CD9, CD63, CD81, Tsg101, Alix, Flotillin-2, and Lamp-1.



Related products

Q28: Are antibodies for exosome marker detection by Western Blotting available from Wako?

We distribute the following antibodies successfully used for this purpose at our Laboratories.

Antigen Reactivity Antibody Manufacturer Use
CD63 human Mouse anti-CD63 monoclonal antibody (3-13) Wako, Code: 012-27063 WB、ELISA
CD81 human Mouse anti-CD81 monoclonal antibody (1D6) Novus, Code:NB100-65805(Wako 559-30131 WB
human Mouse anti-CD81 monoclonal antibody (M38) Novus, Code:NBP1-44861(Wako 550-30161 ELISA
TSG101 human Mouse anti-TSG101 monoclonal antibody (4A10) Novus, Code:NB200-112(Wako 553-30151 WB
Alix human Mouse anti-Alix monoclonal antibody (3A9) Novus, Code:NB100-65678(Wako 552-30121 WB


Q29: Is a kit for purification of RNA from the purified extracellular vesicles available from Wako?

Yes. Our microRNA Extractor SP Kit (Code: 295-71701) are capable of purifying microRNA and mRNA more efficiently than the AGPC method.



Q30: Is a magnetic stand available from Wako?

We distribute Magnetic stand (Code: 290-35591).



Experimental conditions

Q31: Tell me the conditions for exosome secretion enhancement with monensin sodium salt.

The final concentration of monensin sodium salt used for culture of K562 cells is 10µM. Monensin sodium salt is dissolved in ethanol to make a concentration of 10 mM and 1/1000 volume of this solution is added to the culture medium.

Reference
Exosome Release Is Regulated by a Calcium-dependent Mechanism in K562 Cells.
J Biol Chem., 2003 May 30, 278 (22), 20083-90.



Q32: Could you tell me how to prepare a positive control sample?

Culture control cells such as HEK293 or HEK293T (see below for details) and collect approximately 20 mL of culture supernatant. Then concentrate the culture supernatant to 1 mL and purify exosomes according to the instruction manual. This positive control is used to identify expression of exosome markers CD9, CD63, and CD81.
Culture the cells in a serum-supplemented medium for one day, change to a serumfree medium, and grow the cells for additional 3 days.



Q33: Tell me the protocol for BCA assay.

A calibration curve for standards is prepared according to the following protocol. Since protein concentrations of purified exosomes are rather low, use undiluted samples for BCA protein assay.

(1) Pipette 25 µL per well of standard BSA solutions (250, 125, 62.5, 31.25, 15.625 µg/mL) and a standard BLANK into a 96-well plate.
(2) Pipette 25 µL per well of a purified exosome preparation and Elution Buffer (BLANK) on a 96-well plate.
(3) Add 200 µL per well of a mixture of Reagent A and Reagent B (A: B=50: 1) of Protein Assay BCA Kit (Code: 297-73101) to each sample-containing well.
(4) Incubated the plate at 60°C for 30 minutes.
(5) Allow the plate to cool at room temperature.
(6) Measure the absorbance at 560 nm.


Troubleshooting

Q34: My purification results are not satisfactory. How should I prepare for a successful purification?

Prepare a positive control in reference to Q32. In addition, enlarge the culture scale as there is possibility that the amount of extracellular vesicles in the culture medium is small.



PS Capture™ Exosome ELISA Kit (Anti Mouse IgG POD)


Kit specifications and performance

Q1: Do I have to prepare a standard for every assay? And do I have to use Wako MagCapture™ Exosome Isolation Kit PS for preparation of the standard?

When you perform a quantitative assay, prepare an extracellular vesicle sample as standard. Although an extracellular vesicle sample purified by ultracentrifugation or polymer precipitation may also be used as standard, an extracellular vesicle sample purified by the PS affinity method based on the principle identical with that of the assay is recommended (see the instruction manual included in the kit for details of the preparation method).



Q2: Why does this kit include no standard?

The standard and assay samples must be derived from identical cell species, because the type and amounts of surface marker proteins on extracellular vesicles secreted may vary depending on the cell type. Therefore, this kit does not include a standard. Prepare a standard purified from culture supernatant of cells identical with the source cells of assay samples.



Q3: Is this kit compatible with direct assay of serum and plasma samples?

This kit is not recommended for direct assay of serum and plasma samples from human, mouse, and rat, because the secondary antibody for detection included in it reacts with human, mouse, and rat IgG non-specifically. However, this kit is compatible with qualitative assay of extracellular vesicle samples purified from serum and plasma specimens using MagCapture™ Exosome Isolation Kit PS. It is also compatible with qualitative assay of extracellular vesicles purified by ultracentrifugation or polymer precipitation.



Q4: Is this kit compatible with direct assay of cell culture supernatant samples?

This kit is compatible with direct assay of cell culture supernatant from both serum-free and FBS-supplemented media because primary antibody (Anti- CD63 antibody) and secondary antibody included in the kit don’t react with bovine IgG non-specifically. Utilize this kit for both quantitative and qualitative analyses of extracellular vesicles in cell culture supernatant samples.



Q5: Can I replace the primary antibody with another antibody?

Yes. Choose a mouse antibody against the surface marker you want to detect and investigate the optimal concentration according to the instruction manual.



Q6: How can I store the remaining reagents?

See [6. The storage method of each reagent when the kit is separately used] in the instruction manual included in the kit.



Comparison with conventional methods

Q7: Is the detection sensitivity of this kit higher than other methods?

This kit has been confirmed to detect extracellular vesicles at a sensitivity higher than those of ELISA methods involving antibody immobilization or direct immobilization of purified samples on the plate. In addition, a correlation between ELISA results obtained with this kit and Western Blotting results has been confirmed.



Operational procedures and composition of the kit

Q8: How long is the operation time for ELISA using this kit?

The entire kit process takes approximately 5 hours, including immobilization of diluted cell culture supernatant specimens or purified and diluted extracellular vesicle samples onto a 96-well plate for 2 hours, reaction with the primary antibody for 1 hour, reaction with the secondary antibody for 1 hour, and reaction with tetramethylbenzidine (TMB) for 30 minutes. With washing and other operations included, the assay is completed in approximately 5 hours. After addition of Stop Solution, measure the absorbance at the main wavelength 450 nm and the complementary wavelength 620 nm (600 - 650 nm).



Q9: Is an Exosome Capture 96 Well Plate compatible with recycling?

No. An Exosome Capture 96 Well Plate is not compatible with recycling by regeneration, because Stop Solution denatures proteins on the plate.



Q10: Is there any step that can be carried over to next day?

Immobilization of individual samples onto the plate may be prolonged up to overnight at 4°C without any problem.



Sample amount/volume

Q11: How much is the minimum sample amount required for detection using this kit?

Extracellular vesicles corresponding to 1 ng protein are detectable using this kit. The detection limit of extracellular vesicles purified from COLO201 cell culture supernatant was 11 pg (it has been confirmed that the detection limit varies depending on the cell strain).



Q12: How much sample volume is required for direct assay of culture supernatant?

Culture supernatant of a few µL in volume (1-5 µL) is sufficient for assay. This is recommended for monitoring of change in number of extracellular vesicles in culture medium over time and assay of new cell culture supernatant. However, the number of extracellular vesicles in culture medium may be limited depending on the cell species (e.g., iPS cells) and the sample volume per well used for ELISA should be investigated as necessary. If the number of extracellular vesicles in the sample is unknown, use of undiluted culture supernatant samples (100 µL) is recommended.



Related products

Q13: Are there any primary antibodies recommended for detection?

The following antibodies have been confirmed to be compatible with ELISA at our Laboratories.

Antigen Reactivity Antibody Manufacturer Use
CD63 human Mouse anti-CD63 monoclonal antibody (3-13) Wako, Code: 012-27063 WB、ELISA
CD81 human Mouse anti-CD81 monoclonal antibody (M38) Novus, Code:NBP1-44861
(Wako 550-30161
ELISA
CD9 Mouse Rat anti-CD9 monoclonal antibody (MZ3) Bio Legend, Code:124802 ELISA
CD63 Mouse Rat anti-CD63 monoclonal antibody (NVG-2) Bio Legend, Code:143902 ELISA
CD81 Mouse Armenian hamster anti-CD81 monoclonal antibody (Eat-2) Bio Legend, Code:104902 ELISA

Trouble shooting

Q14: My detection results are not satisfactory. Could you tell me what to check for?

Check if any of the reagents has been expired. When you fail to detect a positive signal even with Control Primary Antibody Anti CD63 (100 × ) (included in this kit), it may be ascribable to an expression level of CD63 under the detection limit or some other cause. Please inquire to us in such a case.


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