PS Capture™ Exosome Flow Cytometry Kit

Features

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

Sample Type·Kit Contents

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

Kit Components (300 assays)	Amount
Exosome Capture Beads	1 x 3 mL
Washing Buffer (10×)	2 x 45 mL
Exosome Binding Enhancer (100×)	1 x 15 mL

Princeple

Qualitative analysis of exosomes in cell culture supernatant of K562 cell line

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.

Sample	Cell culture supernatant of K562 cells: 33 μL/Assay
Detection antibody	PE-anti-CD63 (BD Biosciences, 556020) PE-anti-CD9 (Novus Biologicals, NB100-77915PE) PE-anti-CD81 (Novus Biologicals, NBP1-44861PE)

Qualitative analysis of exosomes in serum and plasma

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.

Sample: 33 μL/Assay each	Human serum Human heparin plasma Human EDTA plasma (buffer exchanged)
Detection antibody	PE-labeled anti-CD9 antibody (Novus Biologicals, NB100-77915PE)

Flowchart of Sample Preparation

Cell culture supernatant

Serum・Heparin plasma

EDTA plasma and Citrated plasma

Pretreatment method of 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”.

Recommended reaction scale

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.

Qty of Reaction	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

Outline of Procedure ~Basic Protocol for 2 reactions~