PS Capture™ Exosome Flow Cytometry Kit
Extracellular vesicles (EVs), including exosomes, are known to be heterogeneous, and flow cytometry analysis is expected to be a valuable method for analyzing the characteristics and composition of exosomes. However, the diameter of exosomes is around 100 nm, which presents a challenge for analysis under conditions typically designed for cell sizes on the μm scale.
Fujifilm Wako developed the PS Capture™ Exosome Flow Cytometry Kit which is designed for highly sensitive detection of EVs with maker proteins of interest by flow cytometry. It immobilizes EVs on magnetic beads using the PS affinity method, which utilizes phosphatidylserine-specific binding protein Tim4 and magnetic beads.
Product Overview
PS Capture™ Exosome Flow Cytometry Kit is designed for highly sensitive detection of EVs, including exosomes, with maker proteins of interest by flow cytometry. It immobilizes EVs on magnetic beads using the PS affinity method, which utilizes phosphatidylserine-specific binding protein Tim4 and magnetic beads.
Features
- Highly sensitive qualitative analysis by flow cytometry
- Easy operation with magnetic beads
- No purification necessary; samples can be directly measured
- Assay completes in 3 hours from isolation to staining
Sample (Recommended)
- Cell culture supernatant
- Serum
- Plasma (EDTA/Heparin)
Principle
The PS affinity method, Fujifilm Wako’s proprietary EV isolation technique, utilizes the Tim4 protein, which specifically binds to phosphatidylserine (PS) present on the surface of EVs1). The high specificity of the PS-Tim4 interaction enables to capture high-purity EVs in their intact state.
(1) Isolation of EVs
EVs are isolated from the sample by Exosome Capture Beads.
(2) Staining of EVs
EVs are stained with fluorescent-labeled anti-EV marker antibodies.
(3) Flow cytometry analysis
Flow cytometry analysis is performed with EVs bound to magnetic beads.
Protocol
Number of Reactions and Sample Volume
In the EV isolation process, a standard protocol is set for 1.5 mL microtubes (for two reactions). To increase the scale of reactions, the amount of Exosome Capture Beads and sample volume should be scaled up according to the table below.
| Number of Reactions | Exosome Capture Beads (μL) |
Sample Volume (μL) |
|---|---|---|
| 2 reactions (basic) | 30 | 100 |
| 3 reactions | 40 | 133 |
| 4 reactions | 50 | 167 |
| 5 reactions | 60 | 200 |
| 6 reactions | 70 | 233 |
| 7 reactions | 80 | 267 |
| 8 reactions | 90 | 300 |
| 9 reactions | 100 | 333 |
| 10 reactions | 110 | 367 |
Data
Analysis of Surface Antigens of EVs in Cell Culture Supernatants
EVs in K562 cell culture supernatants were immobilized this kit and with other companies' products (anti-CD9, CD63, and CD81 antibody-coated magnetic beads), and fluorescent-labeled anti-CD9, CD63, and CD81 antibodies were bound for analysis of EV surface antigen by flow cytometry.
Exosome Flow Cytometry Kit
magnetic beads
magnetic beads
magnetic beads
- Isotype Control
- PE labeled anti-CD9 antibody
(Novus Biologicals, NB100-77915PE) - PE labeled anti-CD63 antibody
(BD Biosciences, 556020) - PE labeled anti-CD81 antibody
(Novus Biologicals, NBP1-44861PE)
Reference Data －Comparison of Signal/Background Ratios－
(1) PS Capture™ Exosome Flow Cytometry Kit
(2) Competitor’s product: Anti-CD81 antibody-coated magnetic beads
(3) Competitor’s product: Anti-CD9 antibody-coated magnetic beads
(4) Competitor’s product: Anti-CD63 antibody-coated magnetic beads
[Result]
The PS Capture™ Exosome Flow Cytometry Kit was able to detect EV surface antigens with high sensitivity.
Analysis of Surface Antigens of EV in Serum and Plasma
EVs contained in 100 μL of human serum, heparin plasma, and EDTA plasma (buffer-exchanged) were immobilized onto magnetic beads. Detection of EVs was performed using PE-labeled anti-mouse IgG isotype control and PE-labeled anti-human CD9 antibody.
- PE-labeled mouse IgG isotype control
- PE-labeled Anti-CD9 Antibody
(Novus Biologicals, NB100-77915PE)
[Result]
Using this product, CD9 in human serum and plasma (heparin/EDTA) was detected.
Detection of proteins inside EVs by Flow Cytometry
EVs purified from HEK293T cell culture supernatant were permeabilized with EV-Perm™ Permeabilization Pretreatment Kit for Exosome Membrane (Product No. 294-85701) , and Alix that is the protein inside EVs, and CD63 that is EV surface marker protein were detected using this kit.
[Result]
With permeabilization, Alix inside EVs was detected by flow cytometry.
About commercial or for-profit purposes
Please use this product for research purposes only. If you wish to use it for commercial or for-profit purposes, please contact Fujifilm Wako at ffwk-labchem-tec@fujifilm.com.
References
- Nakai, W. et al.: Sci. Rep., 6(1), 1(2016).
A novel affinity-based method for the isolation of highly purified extracellular vesicles
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For research use or further manufacturing use only. Not for use in diagnostic procedures.
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