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Home  > Products >  Life Science > Genetic Research  >  MagCapture™ Exosome Isolation Kit PS

Exosome isolation by novel affinity molecule
MagCapture™
Exosome Isolation Kit PS

  Exosomes and other extracellular vesicles (EVs) are small membrane vesicles containing protein, mRNA, micro RNA, DNA and lipids, which are secreted by various cells and are stable in body fluids including blood, saliva, urine, cerebrospinal fluid (CSF) and breast milk. These EVs have been recognized as messengers of cell-to-cell communication, and biomarkers for various diseases. Conventionally, ultracentrifugation, affinity purification using antibody to surface antigen, and precipitation with polymer reagents are used for isolation of exosomes. However, these methods are not fully satisficing in all performances criteria such as recovery efficiency, purity and operability.
   MagCapture™ Exosome Isolation Kit PS adopts a novel affinity purification method using magnetic beads and phosphatidylserine (PS)-binding protein (PS affinity method). This kit can easily isolate high purity exosomes and other EVs from cell culture medium and body fluids at high yield by a normal microcentrifuge. If higher purity exosomes are needed, please use the 10,000 x g supernatant as sample. This kit enables the isolation of exosomes and other EVs as intact forms because the captured EVs are eluted from magnetic beads with metal-chelating reagent at neutral pH. The isolated intact exosomes and other EVs can be used for various applications including electron microscopic analysis, nanoparticle tracking analysis, administration of EVs and analysis of molecular constituents such as proteins, lipids, or nucleic acids.



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1. Affinity method for phosphatidylserine (PS) on membrane surface

A novel affinity-based method for phosphatidylserine (PS) on the surface of extracellular vesicles

  Extracellular vesicles are captured by phosphatidylserine (PS)-binding proteins and metal ions, then captured extracellular vesicles are eluted with a chelating reagent.

2. Features

High purity exosomes can be easily isolated by PS affinity method.

 

Novel affinity method

· Isolating by a PS-binding molecule
· Low background
· Mild elution by a chelating reagent under a neutral pH condition


High purity and intact exosomes

No required ultracentrifugation

· Easy operation using magnetic beads
· Optimized protocol

High reproducibility


Comparison with other purification method
Methods Exosome purity Recovery
amount
Intact or not
MagCapture™ (PS affinity method) ■■■ ■■■
Yes
Ultracentrifugation ■■ ■■
Yes
Polymer-based precipitation ■■■
Yes
Exosome surface antigen affinity method
(using the antibody)
■■■ ■■
No

Target samples: cell culture supernatant, serum, plasma, urine, etc.

3. Product Lists

Product Name Package Size Wako Cat. No. Storage
MagCapture™ Exosome Isolation Kit PS 2 purification 299-77603 Keep at 2-10°C
  [Kit Contents]
    (1) Streptavidin Magnetic Beads
    (2) Biotin-labeled Exosome Capture
    (3) Exosome Capture Immobilizing Buffer
    (4) Exosome Binding Enhancer (x 500)
    (5) Washing Buffer
    (6) Exosome Elution Buffer
    (7) Reaction Tubes

120 μL x 1 tube
20 μL x 1 tube
7 mL x 1 bottle
100 μL x 1 tube
30 mL x 1 bottles
1 mL x 1 bottle
4 tubes
MagCapture™ Exosome Isolation Kit PS 10 purification 293-77601 Keep at 2-10°C
  [Kit Contents]
    (1) Streptavidin Magnetic Beads
    (2) Biotin-labeled Exosome Capture
    (3) Exosome Capture Immobilizing Buffer
    (4) Exosome Binding Enhancer (x 500)
    (5) Washing Buffer
    (6) Exosome Elution Buffer
    (7) Reaction Tubes

600 μL x 1 tube
100 μL x 1 tube
35 mL x 1 bottle
500 μL x 1 tube
75 mL x 2 bottles
5 mL x 1 bottle
22 tubes

※Used Exosome Capture-immobilized beads can be recycled for “Repeated extraction of extracellular vesicles from the same sample” and “Purification of extracellular vesicles from culture supernatant sample of the same lot and body fluid sample of the same lot”. Recycling is up to 4 times.

4. Analysis of extracellular vesicles purified from serum / plasma / urine samples

Comparing the yield of exosomes isolated from human normal serum

  Exosomes were isolated from normal human serum by using MagCapture™, ultracentrifugation and affinity method with antibody against surface antigen of exosome, followed by Western Blot with the anti-CD9 and anti-CD63 antibodies.

Lane 1: Ultracentrifugation
Lane 2: MagCapture™
Lane 3: Exosome Isolation reagent with antibody against CD9 [Supplier A]
Lane 4: Exosome Isolation reagent with antibody against CD63 [Supplier A]
Lane 5: Exosome Isolation reagent with antibody against CD81 [Supplier A]
Lane 6: Exosome Isolation reagent with antibodies [Supplier A]
(antibody beads-mixture of CD9, CD63, CD81 & EpCAM)
■ Detection antibody
Anti-CD9 rabbit pAb, System Bioscience
Anti-CD63 rabbit pAb, System Bioscience
The yield of exosomes by MagCapture™ is higher than ultracentrifugation and the antibody-based method.
■ About data of WB
Each signal corresponds to 150μL of the serum sample. 15μL of eluate and 5μL of 4×SDS sample buffer were mixed and applied all in each well.

 

Isolating exosomes from human EDTA-plasma

  Exosomes were isolated from EDTA-plasma, EDTA-plasma (buffer-exchanged), and serum by using MagCapture™, followed by Western Blot with the anti-Flotillin-2 antibody.

1: EDTA-plasma
2: EDTA-plasma (buffer-exchanged)
3: Serum
■ Detection antibody
  Anti-Flotillin-2 mouse mAb (29/Flotillin-2), BD Bioscience
  ■ Sample volume
  1 mL each
By exchanging buffer by ultrafiltration, exosomes can be isolated more efficiently.
■ About data of WB
  Each signal corresponds to 150μL of various samples.
  15μL of eluate and 5μL of 4×SDS sample buffer were
  mixed and applied all in each well.

 

Comparing the yield of exosomes isolated from human normal urine

  Exosomes were isolated from normal human urine by using MagCaptureTM, ultracentrifugation and polymerbased precipitation, followed by Western Blot with the anti-CD9 antibody.

 

Comparison of recovery amount of marker protein   Comparison of NTA  
■ Detection antibody Anti-CD9 rabbit pAb, System Bioscience
High purity exosomes can be isolated from urine sample.
■ Sample volume
1 mL each
■ About data of WB
Each signal corresponds to 150μL of the urine sample. 15μL of eluate and 5μL
of 4×SDS sample buffer were mixed and applied all in each well.

5. Analysis of extracellular vesicles purified from culture supernatant samples

  The yield and purity were compared for exosomes isolated from K562 (human chronic mylogenous leukemia: CML) cell culture supernatants (serum-free medium, or 10% Exosome-depleted FBS medium) by using MagCapture™, ultracentrifugation and polymer-based precipitation method.


〈Sample Preparation Method〉
MagCaptureTM Exosome Isolation Kit PS
  Exosomes were recovered from 1 mL of pretreated (10,000×g, 30 min.) cell culture supernatant of K562 cells (serum-free medium or 10% exosome-depleted FBS ※ 1 included medium) by using PS affinity method's standard protocol (reaction time: 3 hours)

Ultracentrifugation
  10 mL of pretreated (10,000×g, 30 min.) cell culture supernatant of K562 cells (serum-free medium or 10% exosome-depleted FBS ※ 1 included medium) were ultracentrifuged at 110,000×g, 70 min., and the precipitates were suspended with TBS. Then, the suspension was ultracentrifuged again and the precipitated pellet was recovered as exosome sample.

Polymer-based precipitation
  Exosomes were collected from 1 mL of pretreated (10,000×g, 30 min.) cell culture supernatant of K562 cells (serum-free medium or 10% exosome-depleted FBS※1 included medium) by Supplier A’s product in accordance with manual (Precipitation time: overnight).

※1・・・Centrifuged for 2 hours at 110,000 × g, and the supernatant was collected so as not to take up the precipitate.

5-1. Electron microscopic analysis and Nano analysis of isolated exosomes

  The particle size of exosomes from K562 cell culture supernatant (serum-free medium) using MagCapture™, ultracentrifugation and polymer-based precipitation, respectively was determined by using NanoSight LM-10. The collected exosomes (2-4 x 1010 particles) were fixed by 2% paraformaldehyde and analyzed by electron microscopy.

MagCapture™ collected many particles around 50-100 nm in size. With electron microscopy, numerous extracellular vesicles can be seen.
5-2. Comparing the recovery amount and purity of exosomes (serum-free)

  Exosomes were collected from cell culture supernatant of K562 cells (serum-free medium) by MagCaptur™, ultracentrifugation and polymer-based precipitation. The recovery amount and purity were analyzed by silver staining and Western Blot with anti-CD63, anti-Flotillin-2, and anti-Lamp-1 antibodies.

Comparison of recovery amount
(Recovery amount from 150 μL cell culture supernatant)
Comparison of purity
(Amount of marker proteins per 200 ng of total protein)
■ Detection antibody
Anti-CD63 mouse mAb
(MEM-259)

Anti-Flotillin-2 mouse mAb
(29/Flotillin-2), BD Bioscience

Anti-Lamp-1 mouse mAb
(25/Lamp-1), BD Bioscience
Lane 1: MagCaptureT™
Lane 2: Ultracenrifugation
Lane 3: Polymer-based precipitation[Supplier A]
With MagCapture™, the recovery performance of exosomes is excellent and the amount of contaminant of proteins is very low, thus the balance of purity and recovery amount is the best.
5-3. Comparing the recovery amount and purity of exosomes (10% exosome-depleted FBS)

  The exosomes were collected from cell culture supernatant of K562 cells (10% exosome-depleted FBS included medium) by MagCapture™, ultracentrifugation and polymer-based precipitation. The recovery amount and purity were analyzed by silver staining and Western Blot with anti-CD63, anti-Lamp-1, and anti-Flotillin-2 antibodies. Furthermore, collected samples by each method were analyzed by mass spectrometry and compared the percentage of human-derived peptides from K562 cells.

Comparison of recovery amount
(Recovery amount from 150 μL cell culture supernatant)
Comparison of purity
(Amount of marker proteins per 200 ng of total protein)
■ Detection antibody

Anti-CD63 mouse mAb
(MEM-259)

Anti-Flotillin-2 mouse mAb
(29/Flotillin-2), BD Bioscience

Anti-Lamp-1 mouse mAb
(25/Lamp-1), BD Bioscience
  Lane 1: MagCapture™
Lane 2: Ultracentrifugation
Lane 3: Polymer-based precipitation[Supplier A]
 

 

Comparing the percentage of human-derived peptides identified by MASS analysis  

With MagCapture™, high purity exosomes are recovered even from culture medium with FBS, thus MASS analysis can be done with low background.

The ratio of human-derived peptides (%) MASS analysis data was provided by Dr. R. Hanayama at Graduate School of Medicine, Kanazawa University and Dr. W. Nakai at iFReC Osaka University.
5-4. Comparison of microRNA and mRNA recovery amount from exosomes derived from normal human serum

Experimental data

(1) Comparison of microRNA and mRNA recovery amount from exosomes prepared by different techniques

  After isolation of exosomes from normal human serum samples by ultracentrifugation and the PS affinity method, RNA was recovered using microRNA Extractor SP Kit (Code No. 295-71701). microRNA (let-7a, miR-16, miR-92a, miR-142-3p) and mRNA (GAPDH, PIK3CB) were determined by quantitative PCR and compared it using Ct values.

   ■ Amounts of individual microRNA species    ■ Amounts of individual mRNA species
microRNA and mRNA were recovered more efficiently from exosomes isolated by the PS affinity method than those isolated by ultracentrifugation.

 

(2) Comparison of RNA extraction methods from the PS affinity fractions

  After isolation of exosomes from normal human serum samples by the PS affinity method, RNA was recovered using microRNA Extractor SP Kit (Code No. 295-71701) or acid guanidinium thiocyanate-phenolchloroform extraction (AGPC method). microRNA (let-7a, miR-16, miR-92a, miR-142-3p) and mRNA (GAPDH, PIK3CB) were determined by quantitative PCR and compared it using Ct values.

■ Amounts of individual microRNA species ■ Amounts of individual mRNA species
microRNA and mRNA were recovered more efficiently from exosomes using microRNA Extractor SP Kit than using AGPC method.

Related Product
■ microRNA Extractor SP Kit

  This kit is intended for extraction of total RNA including microRNA from human and animal serum/plasma. It extracts microRNA at a high efficiency without using the deleterious substance such as phenol and chloroform that were indispensable for conventional methods for RNA extraction.

Product Name Pkg. Size Wako Cat. No. Storage
microRNA Extractor SP Kit 50 purifications 295-71701 Keep at 2-10°C
5-5. Proteomic Analysis of Exosomes

[Description and results of experiment]
  Exosomes were purified from K562 cell culture supernatant containing 10% exosome-free FBS using either the PS affinity method, ultracentrifugation, or polymer precipitation. The obtained exosome samples were separated by 10% polyacrylamide gel electrophoresis and the individual whole protein bands were cut out. After in-gel digestion, proteins were identified by liquid chromatography mass spectrometry (LC-MS). Proteins identified in exosome preparations purified by the 3 different methods (n=3 for each method) were also compared for Pair-wise correlations.

Comparison of the top 10 proteins identified by MASS analysis

White columns: human protein derived from EVs
Gray columns: bovine protein contaminants derived from FBS
Red color: marker proteins of EVs
Sample:
K562 cell culture Sup.
(10% exosome-depleted FBS included)
PS Affinity Ultracentrifugation Polymer Precipitation
1 Heat shock cognate 71 kDa Protein DNA-dependent protein kinase catalytic subunit Complement C3
2 Annexin A6 Transferrin receptor protein 1 Alpha-2-macroglobulin
3 Transferrin receptor protein 1 Serum albumin Fibronectin
4 V-type proton ATPase catalytic subunit A ATP-dependent RNA helicase A Serum albumin
5 Flotiillin-2 Tubulin beta-5 chain Thrombospondin-1
6 Programmed cell death 6-interacting protein Heat shock cognate 71 kDa protein Complement C4
7 4F2 cell-surface antigen heavy chain Fatty acid synthase Alpha-1-antiproteinase
8 Annexin A1 4F2 cell-surface antigen heavy chain Apolipoprotein B-100
9 Kinase D-interacting substrate of 220 kDa U5 small nuclear ribonucleoprotein helicase Hemoglobin fetal subunit beta
10 Annexin A2 Tubulin beta-4B chain Tubulin beta-5 chain
While proteins of bovine serum origin are present in the greater amount of exosome preparations obtained by polymer precipitation, more exosome marker proteins are identified in exosome preparations obtained by the PS affinity method.

Comparison of Pair-wise correlation

Sample:
K562 cell culture Sup.
(10% exosome-depleted FBS included)

 

  Reproducibility
Ultracentrifugation (UC)
Polymer Precipitation
PS Affinity


Polymer precipitation and the PS affinity method exhibit high intra-method correlation, while the intra-method correlation for ultracentrifugation is a little bit lower. Inter-method correlations between different methods are relatively low. The different purification methods might yield different exosome populations.
Sci Rep. 2016 Sep 23;6:33935. Nakai W et al.  
5-6. Assay of protein concentration in exosomes

Assay of protein concentration in exosomes using Protein Assay BCA Kit

  Protein Assay BCA Kit, capable of assaying total protein concentration in solution using bicinchoninic acid (BCA), is the most widely used protein assay kit. It is based on the principle of reduction of Cu2+ to Cu+ by protein under basic conditions. Chelate formation between Cu+ and BCA generates a purple-colored chelate. Since the purple color becomes more intense in proportion with protein concentration, the protein concentration is determined by comparing this absorbance at 562 nm with a standard curve of absorbance from varying bovine serum albumin (BSA).
  Here, the protein concentration in an exosome preparation purified from cell culture supernatant using MagCapture™ Exosome Isolation Kit PS was determined according to the high-sensitivity protocol for Protein Assay BCA Kit.


[Description and results of experiment]
  An exosome solution purified using MagCapture™ Exosome Isolation Kit PS pipetted into a 96-well plate in 25 μL aliquots and then 200μL of a mixture of Reagent A and Reagent B included in Protein Assay BCA Kit was added to each well. After subsequent incubation at 60°C for 30 minutes, the absorbance was measured at 560 nm (Figure 1). The result demonstrated that the protein concentration in a purified exosome preparation was able to determine using the high-sensitivity protocol for Protein Assay BCA Kit.

Figure 1. A BSA calibration curve determined using Protein Assay BCA Kit and assay of protein concentration of a purified exosome preparation

Product Lists
Product Name Pkg. Size Wako Cat. No. Storage
Protein Assay BCA Kit 250 assays 297-73101 Room Temperature
2 mg/mL Albumin Solution from Bovine Serum 1 mL × 10 015-25613 Room Temperature
[Overview of BCA assay protocol]
Since protein concentrations of exosome preparations are rather low, use undiluted samples for BCA protein assay.
(1) Pipette 25μL per well of standard BSA solutions (250, 125, 62.5, 31.25, 15.625μg/mL) and a standard BLANK into a 96-well plate.
(2) Pipette 25μL per well of a purified exosome preparation and Elution Buffer (BLANK) on 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) Incubated the plate at 60℃ for 30 minutes.
(5) Allow the plate to cool at room temperature.
(6) Measure the absorbance at 560 nm.
5-7. Exosome labeling and uptake assay with HeLa cells

[Experiment overview]
Exosomes purified using MagCaptureTM Exosome Isolation Kit PS were labeled with PKH67 (Sigma) and confirmed the uptake by HeLa cells.
<Exosome labeling with PKH67>
1. Purify exosomes using MagCaptureTM Exosome Isolation Kit PS (from K562 cell culture supernatant on the day of experiment).
2. Determine the protein concentration and particle concentration by BCA Assay and using NanoSight.
3. Dispense the exosome sample solution corresponding to 5μg protein* in a 1.5 mL tube.
  * Prepare an appropriate amount as needed for the experiment.
4. Dissolve 2.0μL PKH67 linker in 0.25 mL of Diluent C (provided with the PKH67 kit) - 4×Dye solution*
  * Prepare an appropriate amount as needed for the experiment.
5. Add 1/3 volume of 4×Dye solution to the exosome sample, mix, and incubate the mixture at room temperature for 5-10 minutes.
6. Equilibrate Exosome Spin Columns (MW 3000) (Thermo #4484449) with PBS according to the protocol provided with the product.
7. Apply 100μL each of exosome sample to each spin column equilibrated as described above* and centrifuge the column to separate unbound dye from labeled exosomes (maximum volume applied: 100μL per column).
  * Prepare as many columns as needed.
8. Add the solution containing labeled exosomes* to HeLa cells seeded on a dish on a preceding day. After 24 hours, perform microscopic observation and flow cytometry.
  * Adjust the number of exosomes added.


Results of microscopic observation Results of flow cytometry
Figure 1 Observation of the uptake of PKH-labeled exosomes into HeLa cells
Exosomes were purified from 4 mL of K562 cell culture supernatant (concentrated from 16 mL of original culture supernatant by ultrafiltration) using the PS affinity method.
• Protein concentration of purified exosomes: 22.3 ng/μL
• Particle concentration: 1.2 × 108 particles/mL
All exosome solution labeled as described above were added to HeLa cells which were seeded on the previous day, and the uptake result was detected by fluorescent microscopy (left) and flow cytometry (right) 24 hours later.
Opti-MEM® : An equivalent amount was labeled with PKH as a negative control and added to cells.
Endocytotic uptake of PKH67-labeled exosomes is confirmed by both microscopy and flow cytometry.

6. Preprocessing protocols for various samples

  This is the section to prepare samples. When exosomes and other large EVs (microvesicles) are needed, prepare 1,200 × g supernatant ※1 as a sample. Additionally, when highly purified exosomes are needed, prepare 10,000 × g supernatant ※2 as a sample. This protocol for sample preparation is set for cell culture medium, serum, and plasma. When other body fluids are used, please examine the appropriate preprocessing protocol by referring to the protocol for serum and plasma.


Cell Culture Medium

 

Serum and Heparin Plasma

EDTA Plasma

 

Protocol for buffer exchange (ultrafiltration)

Perform buffer exchange of 1 mL centrifuged EDTA plasma sample with 50 mL of TBS buffer.

1. Add 19 mL of TBS to Vivaspin20 (100K).
2. Add the 1 mL of centrifuged EDTA plasma sup. to the Vivaspin20 (100K) of 1. and mix (solution A).
3. Centrifuge solution A at 4°C.  (Refer to centrifugal condition in the manufacturer’s instruction manual.)
4. Add 10 mL of TBS to solution A when the upper liquid volume is dropped (solution B).
5. Centrifuge solution B at 4°C.
6. Add 10 mL of TBS to solution B when the upper liquid volume is dropped (solution C).
7. Centrifuge solution C at 4°C.
8. Add 11 mL of TBS to solution C when the upper liquid volume is dropped.
9. Centrifuge solution C at 4°C until the volume becomes under 1 mL.
10. Proceed to “Isolation step”.

 

※1 When exosomes and large EVs are needed, use 1,200 × g sup. fraction as sample.
※2 When exosomes are needed, use 10,000 × g sup. fraction as sample.
※3 When Large EVs are needed, use ppt of Large EVs obtained by centrifugation at 10,000 x g as sample after suspending it with TBS.

※4 The concentration step is an option when using a large volume (~ 50 mL) of cell culture supernatant as a sample for purification. However, since recovery efficiency improves, please perform it as much as possible.

Note : a volume of large or small sample
■In the case of large volume
  The concentration of the sample is recommended when using a large volume (~50 mL) of cell culture supernatant as a sample for purification. Since recovery efficiency improves, please perform it as much as possible.

■In the case of small volume
  Add the appropriate volume of TBS into samples to reach the volume of 0.5 mL to obtain the better mixture of the Exosome Capture-immobilized beads with the sample. (Example: 100-200 μL → 500 μL)

7. Outline of Procedure

8. Reference

W. Nakai, T. Yoshida, D. Diez, Y. Miyatake, T. Nishibu, N. Imawaka, K. Naruse, Y. Sadamura & R. Hanayama, Sci Rep 6, 33935 (2016).
S. Osawa, et al., Biochem. Biophys. Res. Commun., 488(1), 232-238 (2017).
S. Nagashima, et al., J. Virol., 91(22), Published Online (2017).
T. Yoshida, et al., Curr. Protoc. Cell Biol., Published Online (2017).
S. Saito, et al., Sci Rep 8, 3997 (2018).

9. Related Products

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  These products are fusion proteins which were consisted of the extracellular domain of human or mouse TIM family (TIMD1, TIMD3, or TIMD4) and the Fc region of human IgG1 or mouse IgG2a. The Fc region contains a hinge sequence. The TIM (T cell/transmembrane, immunoglobulin and mucin) family plays a critical role in regulating immune responses, including allergy, asthma, transplant tolerance, autoimmunity and the response to viral infections. The unique structure of TIM immunoglobulin variable region domains allows highly specific recognition of phosphatidylserine (PtdSer), exposed on the surface of apoptotic cells.

Product Name Pkg. Size Wako Cat. No. Grade Storage
Human TIM1/Human Fc Chimera, recombinant 100 μg 080-10231 for Genetic Research Keep at -20°C.
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Control Protein (human IgG1-Fc, mouse IgG2a-Fc)

  These products are recombinant proteins of Fc region of human immunoglobulin G1 or mouse immunoglobulin G2a. It should be used as the control protein for the western blotting or the activity test of human Fc or mouse Fc chimeric proteins. These products are lyophilized from 0.2μm-filtered solution in PBS. The non-lytic type has mutations to the complement (C1q) and FcgR I binding sites of the IgG Fc fragment. These mutations render the proteins incapable of antibody directed cytotoxicity (ADCC) and complement directed cytotoxicity (CDC).

Product Name Pkg. Size Wako Cat. No. Grade Storage
IgG1 Fc, Human, recombinant 100 μg 098-07141 for Genetic Research Keep at -20°C
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IgG2a Fc (non-lytic), Mouse, recombinant 100 μg 099-07171

A novel tool for qualitative and quantitative analysis of extracellular vesicles in sample of cell culture supernatant

Product Name Pkg. Size Wako Cat. No. Grade Storage
PS Capture™ Exosome ELISA Kit
(Anti Mouse IgG POD)
96 tests 297-79201 for Genetic Research Keep at 2-10°C
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