Q&As and Troubleshooting

Kit specifications and performance

Q1: What is the principle of this purification method?

This method 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 distri-bution: 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. However, 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 small particle size (Small EVs) including exosomes, use the supernatant obtained after centrifugation at 10,000 x g as a sample.

To purify extracellular vesicles with large particle size (large EVs) including microvesicles, then, collect the supernatant obtained after centrifugation at 1,200 x g, centrifuge this supernatant at 10,000 × g to isolate the precipitate, suspend the precipitate with TBS, and use this suspension as a sample.

To purify both vesicles together, use the supernatant obtained after centrifugation at 1,200 x g as sample.

See instruction manual for details of sample pretreatment 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 with a virus-specific antibody. This applies also to purification methods using antibodies against exosome markers located on the envelope (e.g., CD63).

Q6: Is there citation for virus purification?

In the following paper, virus purification using PS affinity method is reported.

“Vesicle-Cloaked Virus Clusters Are Optimal Units for Inter-organismal Viral Transmission”, M. Santiana, et al., Cell Host & Microbe, 24, 208-220 (2018)

Q7: Do all exosomes have PS exposed on the membrane surface?

Although no finding has been obtained that indicates that all exosomes have exposed PS, we have not confirmed samples that could not be detected in our experience (see Purification results and Detection results on Q7) so far.

In the following paper, cryo-electron microscopic analysis and phenotypic analysis of activated platelet-derived extracellular vesicles using immune-gold colloidal particles are reported. In this paper, it is reported that about 75% of extracellular vesicles from activated platelets have PS exposed on the surface.

“Extracellular vesicles from activated platelets: a semiquantitative cryoelectron microscopy and immuno-gold labeling study”, B. Alain R, et al., Platelets, 28 (3), 263-271 (2017)

Q8: 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, urine, and feces. Userreported applications of this kit include exosome purification from cerebrospinal fluid and saliva.

Q9: 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 x 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 x 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

Q10: 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 from samples difficult to precipitate by ultra-centrifugation.

Q11: What are advantages of this kit over the affinity method using antibodies?

While the affinity method with 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. Because the expression level of the marker protein differs depending on the exosome-derived cell compared with the conventional antibody affinity method targeting one 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).

Q12: What are advantages of this kit over polymer precipitation?

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

　

Operational procedures and composition of the kit

Q13: How long is the operation time for exosome purification using this kit?

Sample pretreatment requires approximately 1 hour, while the entire kit process takes 3 and a half hours 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 1 hour, make sure in advance to confirm that this change does not affect experimental results.

Q14: 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 step 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.

Q15: 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) or gel filtration.

Q16: Are magnetic beads with immobilized Exosome Capture compatible with recycling?

Yes. Used magnetic beads are reusable up to 4 times after regeneration to ensure recovery of exosomes remaining 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 case of 10 test kit) 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 instruction manual for details.

Q17: 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 prepared separately (Our experience: usable after 2 years of storage).

Q18: 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 problems.

　

Sample volume

Q19: How much is the minimum sample volume 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. In addition, it is recommended to add EV-Save™ Extracellular Vehicle Blocking Reagent (Code: 058-09261) to the TBS for fill-up.

Q20: 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 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. Since serum samples cannot be concentrated, this kit is compatible with a serum sample volume only up to 1 mL. See instruction manual for details.

In addition, it has been confirmed that concentration by ultrafiltration decrease exosomes due to absorption to containers and filters. When performing concentration by ultrafiltration, add EV-Save™ Extracellular Vehicle Blocking Reagent Kit (Code: 058-09261) to prevent adsorption loss. However, since this product contains a polymer, using as samples for proteomics analysis is not recommended.

Q21: Why is a molecular weight cut off of 100K recommended for ultrafiltration concentration?

We compare 100K, 300K and 1000K ultrafiltration filters and recommend 100K based on the concentration time and results of concentrated exosomes. 10K and 30K can be used, concentration time will be longer. In addition, in the case of a medium containing albumin, the recovery efficiency may decrease because the albumin is concentrated.

　

Analysis after exosome extraction

Q22: What are the components of exosomes?

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

Q23: What kind of downstream application do 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, proteomics analysis, 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.

Q24: Can I use the extracellular vesicles purified with this kit for uptake assay to cells without any pre-treatment?

Because cytotoxic effects of preservatives have been confirmed, prepare PBS buffer containing 2 mM EDTA separately and use it instead of the elution buffer supplied with the kit. For the purified extracellular vesicles sample obtained, sterilize it using a centrifugal filter unit (Millipore Ultrafree - MC, GV 0.22μm, sterile, Catalog No.: UFC30GV0S). After sterilization treatment, use it for uptake assay etc. Even if EV-Save™ is added to prevent adsorption, it can be used as it is.

Q25: How much is the exosome amount required for electron microscopic analysis?

We have an experience of performing electron microscopic analysis using 2-4 x 10¹⁰ exosome particles/mL particle concentration (measured by Nano-Sight LM10).

Q26: How much exosome is required for microarray analysis?

In our experiment, we have been able to perform microarray analysis by extracting RNA from the number of particles below (measured by NanoSight LM10);
COLO201: 4.6 x 10¹⁰ particles
TIG3: 1.7 x 10¹⁰ particles
iPS: 1.9 x 10⁹ particles

Q27: How much exosome is required for proteomics analysis?

For "Isolation / Purification of Exosome" proteomics analysis (W. Nakai, et al., Sci. Rep., 6, 33935, 2016), approximately 1 μg of purified exosomes were used. (It is derived from approximately 1.5 mL of K562 culture supernatant sample. Since this sample promotes exosome secretion with monensin, the amount of exosome is large.)

Q28: How should I store the extracellular vesicles purified with this kit?

Add EV-Save™ and store at 2 〜10 ℃ or -20 ℃. Store at -80 ℃ for long-term storage. Due to cryoprotective effect of EV-Save™ ensures that the purified extracellular vesicles are not broken even when they are stored frozen. However, since it contains a polymer, using this sample for proteomics analysis is not recommended.

Q29: How do I perform western blotting analysis using the recoverd exosome?

At our laboratory, 15 μL of eluate and 5μL of 4 x SDS sample buffer are mixed and applied for SDS-PAGE. All western blotting experiments described in this guidebook are applied under this condition.

　

Exosome markers

Q30: How are the exosomes purified using this kit identified?

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

Q31: What are the marker proteins identified in exosomes by western blotting?

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

　

Related products

Q32: Are exosome marker antibodies for western blotting available from Wako?

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

Antigen	Reactivity	Antibody	Manufacturer	Application
CD63	Human	Anti CD63, Monoclonal Antibody (3-13)	Wako, Code: 012-27063	WB, ELISA, FCM, IP
CD81	Human, Bovine	Anti CD81, Monoclonal Antibody (17B1)	Wako, Code: 011-27773	WB, ELISA, FCM, IP
CD9	Human, Bovine	Anti CD9, Monoclonal Antibody (1K)	Wako, Code: 014-27763	WB, ELISA, FCM, IP
TSG101	Human	Tsg101 Antibody (4A10)	Novus, Code:NB200-112 (Wako 553-30151)	WB
Alix	Human	Alix Antibody (3A9)	Novus, Code:NB100-65678 (Wako 552-30121)	WB

Q33: 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 AGPC method.

Q34: Is a magnetic stand available from Wako?

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

　

Experimental conditions

Q35: What is the conditions for exosome secretion enhancement with monensin sodium salt.

The final concentration of monensin sodium salt used for culture of K562 cells is 10 μM. 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. Exosome Release Is Regulated by a Calcium-dependent Mechanism in K562 Cells., J Biol Chem., 2003 May 30, 278 (22), 20083-90.

Q36: How should I prepare a positive control sample?

Culture any control cells such as HEK293, prepare the required amount of culture supernatant, and purify exosomes using this kit. The purified exosomes show exosome markers such as CD9, CD63, and CD81 in western blotting and ELISA.

At our laboratory, control cells are cultured in serum-supplemented medium for one day, change to a serum-free medium or exosome-depleted serum-containing medium for about 3 days. Then collect the culture supernatant.

Q37: What is the protocol for BCA assay?

A calibration curve for standards is prepared according to the following protocol.Since protein concentrations of exosomes isolated and purified with this kit is low, it is recommended to measure without diluted.

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 purified exosomes and Elution Buffer (BLANK) to a 96-well plate.
3. Add 200μL 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. Incubate the plate for 30 minutes at 60℃ .
5. Allow the plate to cool at room temperature.
6. Measure the absorbance at 560 nm.

　

Troubleshooting

Q38: My purification results are not satisfactory. How should I prepare for a successful purification?

Prepare a positive control in reference to Q36. Enlarge the culture scale as the amount of extracellular vesicles in the culture medium may be small.

Q39: The total protein amount of the exosome sample purified by this kit is smaller than that of exosome sample collected by other methods. What causes it?

Exosome samples collected by other methods contaminated with many impurities, resulting in a large amount of total protein. On the other hand, although the total protein amount of the exosome sample purified with this kit is small, the amount of exosome actually obtained may be larger than that of the others due to the high purity of the sample.

Kit specifications and performance

Q1: Does this kit include antibodies for detection of exosomes?

This kit does not include fluorescent labeled antibodies for detection of exosomes. Additional purchase of any fluorescein-labeled anti-CD63 antibody (Code: 018-27641), red fluorescent-labeled anti-CD63 antibody (Code: 011-27751), or other appropriate fluorescent-labeled antibody is required.

Q2: Is it able to use a fluorescent labeled secondary antibody for detection?

Yes. detection with a fluorescent labeled antibody is possible. After isolation of exosomes, please proceed with the primary antibody reaction, secondary antibody reaction, and flow cytometry analysis in accordance with the following protocol.

1. Mix an unlabeled primary antibody and Exosome Capture Beads, and allow to stand at room temperature for 1 hour. During the incubation, vortex the mixture for about 5 seconds at 20 minutes, 40 minutes, and 1 hour to stir the magnetic beads.
2. Wash the beads 2 times with 300 μL of WB (+ Enhancer).
3. Dilute a PE-labeled secondary antibody (Code: 115-115-164) (Jackson Immuno Research Laboratories) 100-fold with WB (+Enhancer), and add the secondary antibody solution to the magnetic beads in 2).
4. Allow to stand at room temperature for 1 hour. During the incubation vortex the mixture for about 5 seconds at 20 minutes, 40 minutes, and 1 hour to stir the magnetic beads.
5. Wash the beads 3 times with 300 μL of WB (+ Enhancer).
6. Suspend the magnetic beads in 300 μL of WB (+ Enhancer).
7. Perform flow cytometry analysis.

Q3: Can this kit be used for non-human biological species?

Yes. Exosomes from humans, mice, cattle, and monkeys have been successfully detected.

Q4: Is it possible to use MagCaplure™ Exosome Isolation Kit PS (Code: 293-77601) for flow cytometry analysis?

MagCaplure™ Exosome Isolation Kit PS is a kit for purification of exosomes but not for flow cytometry analysis. For PS Capture™ Exosome Flow Cytometry Kit, magnetic beads and washing buffer are optimized for flow cytometry analysis.

　

Operational procedures and composition of the kit

Q5: How long does it take to run an experiment with this kit?

Pretreatment of the samples takes about 1 hour, and the process of this kit takes about 2 hours and 20 minutes. The process consists of a reaction with the sample for 1 hour, staining of exosomes with fluorescent labeled antibody for 1 hour, and washing for 20 minutes.

Q6: Multiple Exosome Capture Beads may bind to an exosome, forming aggregates of magnetic beads. In such case, is an accurate analysis possible?

It is recommended to adjust gating parameters based on a plot of the forward and lateral scattered lights for gating of a singlet bead fraction only and then detect fluorescent signals of the Exosome Capture Beads in the gated fraction. Generally, the singlet bead fraction accounts for 50% to 70% of the sample overall.

Q7: Is there any method to measure purified exosome samples?

It is recommended to dilute the purified exosomes to an appropriate concentration and proceed to 2. Isolation of extracellular vesicles in instruction manual. For dilution of the purified exosomes, it is recommended to use the washing solution after diluting the washing solution (10 x ) 10 fold with ultrapure water. Purified exosomes can be detectable in the concentration range from 125 to 1000 ng/mL. For a detection antibody, Anti CD63, Monoclonal Antibody (3-13), Red Fluorochrome (635) Conjugated (Code: 011-27751) is recommended.

Q8: Detection using about 10 types of antibodies is planned. What amounts of samples and magnetic beads are necessary for such experiment?

Please refer to Table 2 Recommended reaction scale in instruction manual. In the bottom row of Table 2, amounts for 10 reactions are described. Mix 367 μL of the sample with 110 μL of Exosome Capture Beads to isolate exosomes, wash these beads with WB (+Enhancer), suspend these beads in 1100 μL of WB (+Enhancer); and then proceed to 3. Immunostaining of extracellular vesicles in which the suspended beads are dispensed into 100 μL portions.

　

Sample volume

Q9: How much is the minimum sample volume for detection?

About 33 μL of a sample is required. If an amount of extracellular vesicles in a sample is small, concentration by ultrafiltration of a conditioned medium pretreated with centrifugation is recommended to prepare the sample for detection. (recommended filter; Sartorius Vivaspin 20, cut-off molecular weight 100 K, Product code: VS2041)

　

Related products

Q10: Is a magnetic stand available from Wako?

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

　

Troubleshooting

Q11: Magnetic beads are not gathered to the magnetic stand.

The Exosome Capture Beads of this kit are optimized for flow cytometry detection, and thus the concentration of magnetic beads is low. Therefore, magnetic beads gathered by the magnet may be hardly visible. Before the washing operation, it is recommended to keep the tube still on the magnetic stand for at least 1 minute. Discard the washing buffer gently not to suction the magentic beads.

Kit specifications and performance

Q1: Do I have to prepare a standard for every assay? And do I have to use MagCaptureTM 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 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 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: Can this kit measure extracellular vesicles in serum and plasma directly?

PS Capture™ Exosome ELISA Kit (Streptavidin HRP）is available. PS Capture™ Exosome ELISA Kit (Anti Mouse IgG POD) is not recommended for direct assay of serum and plasma samples from human, mouse, and rat, because the secondary antibody for detection included in the kit reacts with human, mouse, and rat IgG non-specifically.

Q4: Can this kit measure extracellular vesicles in cell culture supernatant directly?

Both kits are available. PS Capture™ Exosome ELISA Kit (Anti Mouse IgG POD) 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. In addition, biotin-labeled antibody (anti-CD63 antibody-biotin) and Streptavidin HRP in PS Capture™ Exosome ELISA Kit (Streptavidin HRP) do not react in the same manner. 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. When using PS Capture™ Exosome ELISA Kit (Anti Mouse IgG POD), choose a mouse antibody against the surface marker of interest and investigate the optimal concentration according to instruction manual.

When using PS CaptureTM Exosome ELISA Kit (Streptavidin HRP), select a biotin-labeled antibody for detection of any surface marker, and optimize concentration according to instruction manual.

If a biotin-labeled antibody is not available, label the antibody with Biotin Labeling Kit-SH (Code: 348-90941) or Biotin Labeling Kit-NH2 (Code: 347-90891).

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.

Q7: What kinds of cell lines can I use for this kit?

The following is a list of cell lines successfully isolated and detected by Wako.

Cell Line	Origin	Isolation	ELISA
A549	Human Caucasian lung carcinoma	ー	〇
BxPC-3	Human primary pancreatic adenocarcinoma	ー	〇
COLO201	Human Caucasian colon adenocarcinoma	〇	〇
COS7	Monkey African green kidney	〇	〇
FM3A	Mouse C3H mammary carcinoma	〇	〇
HCT116	Human colon carcinoma	〇	〇
HEK293	Human Embryo Kidney	〇	〇
HEK293T	Human Embryo Kidney	〇	〇
HeLa	Human cervix carcinoma	〇	〇
HPAF Ⅱ	Human pancreatic tumor	ー	〇
HuH-7	Human hepatocellular carcinoma	〇	〇
HUVEC	Human umbilical vein endothelial cell	〇	〇
iPS	induced pluripotent stem cell	〇	〇
K562	Human Caucasian chronic myelogenous leukaemia	〇	〇
LNCaP	Human Caucasian prostate carcinoma	〇	〇
P388D1	Mouse leukemia	〇	〇
Panc-1	Human Caucasian pancreas	ー	〇
RAJI	Human Burkitt's lymphoma	〇	〇
SH-SY5Y	Human neuroblastoma	ー	〇
TIG-3	Normal human diploid cells.	〇	〇
THP-1	Human monocytic leukaemia	〇	〇
U2OS	Human Osteosarcoma	〇	〇
BM-MSC	Human Mesenchymal Stem Cells from Bone Marrow	〇	〇
iCell-MSC	Mesenchymal Stem Cells from iPS-01279	〇	〇

−：No test　〇：Succeeded

Q8: Can the recovery rate of extracellular vesicles be measured with this kit?

Yes, it can. Please refer to "Exosome ELISA kit" page; Comparison of recovery efficiency with polymer precipitation.

　

Comparison with conventional methods

Q9: Is the detection sensitivity of this kit higher than other methods?

This kit has been confirmed to detect extracellular vesicles at sensitivity higher than those of ELISA methods using immobilized antibodies or direct immobilization of purified samples on the plate. In addition, it has been confirmed that there is a correlation with the Western blot method.

　

Operational procedures and composition of the kit

Q10: How long is the operation time of this kit?

The entire process of PS Capture™ Exosome ELISA Kit (Anti Mouse IgG POD） takes approximately 5 hours, including immobilization of 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.

The entire process for PS Capture™ Exosome ELISA Kit (Streptavidin HRP）is approximately 6 hours. The breakdown of the process is 2 hours for immobilizing extracellular vesicle samples on the plate, 1 hour for the primary antibody reaction, 2 hours for the streptavidin-HRP reaction, and 30 minutes for the TMB reaction. It can be measured in about 6 hours including other washing steps.

After addition of Stop Solution, measure the absorbance at the main wavelength 450 nm and the complementary wavelength 620 nm (600 - 650 nm).

Q11: Can I reuse an Exosome Capture 96 Well Plate?

No. An Exosome Capture 96 Well Plate cannot be reused because Stop Solution denatures proteins on the plate.

Q12: 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℃.

　

Sample volume

Q13: 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 (The detection limit varies depending on the cell lines).

Q14: How much sample volume is required for direct assay of culture supernatant and body fluid sample?

Culture supernatant or body fluid sample of a few μL in volume (1-5 μL) is sufficient for assay. This is recommended for monitoring changes in number of extracellular vesicles in culture medium over time and assay of new cell culture supernatant. However, depending on the cell type (iPS cells, etc.), the amount of extracellular vesicles in the medium may be small. If the amount of extracellular vesicles in the sample is unknown, preliminary experiments for an appropriate amount of sample is recommended.

　

Related products

Q15: Is there a recommended primary antibody for detection?

We have confirmed that the following antibodies can be used for ELISA.

Antigen	Reactivity	Antibody	Manufacturer	Application
CD9	Human, Bovine	Anti CD9, Monoclonal Antibody (1K)	Wako, Code: 014-27763	WB, ELISA, FCM, IP
CD63	Human	Anti CD63, Monoclonal Antibody (3-13)	Wako, Code: 012-27063	WB, ELISA, FCM, IP
CD81	Human, Bovine	Anti CD81, Monoclonal Antibody (17B1)	Wako, Code: 011-27773	WB, ELISA, FCM, IP
CD9	Mouse	Rat anti-CD9 monoclonal antibody (MZ3)	Bio Legend, Code:124802	WB, ELISA
CD63	Mouse	Rat amti-CD63 monoclonal antibody (NVG-2)	Bio Legend, Code:143902	WB, ELISA
CD81	Mouse	Armenian hamster anti-CD81 monoclonal antibody (Eat-2)	Bio Legend, Code:104902	WB, ELISA

　

Troubleshooting

Q16: My detection results are not satisfactory. What should I check?

Check if any of the reagents has been expired. Be sure to add Exosome Binding Enhancer (100 x ) to the washing solution. When you fail to detect a positive signal even with Control Primary Antibody Anti CD63 (100 x ) or Control Biotinylated Antibody Anti-CD63 (100 x ) (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.