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

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”.

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~

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.

Reference

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).