The kit can detect any surface marker proteins on extracellular vesicles (EVs) with high sensitivity by flow cytometry after capturing EVs by PS Affinity Method※1. Before use, it is necessary to prepare fluorescence-labeled primary antibody against exosome surface marker protein, or primary antibody and fluorescence-labeled secondary antibody.

1. "A novel affinity-based method for the isolation of highly purified extracellular vesicles", W. Nakai, T. Yoshida, D. Diez, Y. Miyatake, T. Nishibu, N. Imawaka, K. Naruse, Y. Sadamura ＆ R. Hanayama, Sci Rep 6, 33935 (2016).

• High-Sensitive Qualitative Analysis
• Easy Operation by Magnetic Beads
• Direct Detection without Purification
• Total 3 hours ~ from Isolation to Staining ~

• Cell Culture Supernatant
• Serum
• Plasma (Heparin and EDTA)

Exosomes in cell culture supernatant of K562 cells were isolated using PS Capture Exosome Flow Cytometry Kit or each of anti-CD81-, CD9- and CD63-antibody-immobilized magnetic beads (supplier A), followed by flow cytometric analysis of exosome surface antigens after immunostaining with fluorescence-labeled antibodies.

1. PS Capture Exosome Flow Cytometry Kit
2. Anti-CD81 antibody immobilized magnetic beads
3. Anti-CD9 antibody immobilized magnetic beads
4. Anti-CD63 antibody immobilized magnetic beads

Each signal value was normalized using the value of Isotype.

It was confirmed that the PS Capture Exosome FCM Kit can detect the exosome surface antigen with high sensitivity compared with competitors' products, regardless of which detection antibody is used.

Exosomes in human serum and human plasma (EDTA plasma, heparin plasma) were isolated using PS Capture Exosome Flow Cytometry Kit, followed by flow cytometric analysis of exosome surface antigens after immunostaining with PE-labeled mouse IgG isotype control and PE-labeled anti-human CD9 antibody.

In any of the samples, the peak shift of fluorescence intensity was confirmed when stained with PE-labeled anti-human CD9 antibody.

Cell culture supernatant

Serum・Heparin plasma

EDTA plasma and Citrated plasma

EDTA and Citric acid, which is the anticoagulant inhibit the binding of extracellular vesicles to Exosome Capture Beads. Therefore, perform pretreatment with sodium heparin solution.

1. Prepare 1,000 U/mL sodium heparin solution with purified water. [Example of product used: Code No. 085-00134]
2. Add 1 : 200 volume of sodium heparin solution to 100 μL of 10,000 × g supernatant (final concentration : 5 U/mL) and mix.
3. Add 1 : 20 volume of Exosome Binding Enhancer (100×) further and mix.
4. Proceed to “Isolation step”.

Basic protocol is set as 2 reactions using a 1.5 mL microcentrifuge tube to isolate EVs from samples with Exosome Capture Beads.
For scale-up, increase the amount of Exosome Capture Beads and samples. Recommended reaction scale is presented in the right table.
Note: 10 reactions are maximum for one 1.5 mL microcentrifuge tube.