ExoIsolator Exosome Isolation Kit / ExoIsolator Isolation FilterEX10_3 Product Manual

Exosomes, a form of secreted extracellular vesicles, contain various proteins and nucleic acids; therefore, exosomes have various effects on recipient cells¹⁾. Recently, there have been many reports related to exosomes related, for example, metastasis and malignant progression of cancer, therapeutic research, and diagnostic research. Many methods have been developed for the purification of exosomes for research use. Of these, the main method is ultracentrifugation (UC). However, UC requires complicated operations and a long time to collect exosomes.
Dojindo’s ExoIsolator Exosome Isolation Kit can collect exosomes from cell supernatants with a recovery rate equivalent to that of UC. Unlike UC, ExoIsolator Exosome Isolation Kit requires only the filtration procedure, exosomes are obtained quickly without any complicated operations.

Figure 1. Principle of exosomes purification
(Patent pending)

Store at room temperature

• Aspirator
  • Prepare an aspirator as commonly used for cell experiments. Recommended aspiration pressure for use is −25 kPa or lower.
• 0.22 μm filters
• The 0.22 μm filter is used for pre-filtration of the cell supernatant to remove cellular fragments and cell debris.
• Micropipettes
• 1.5 ml microtubes
• Phosphate-buffered saline (PBS)

• The new version of the Isolation Filter is reversible.
• Please make sure the step 4-8 in “General Protocol” below and “Operation Video” on our web site for setting the Isolation Filter on the Filter Holder.
• The Filter Holder should be washed and autoclaved before using. The Filter Holder is not sterilized.
• The Filter Holder is autoclavable and can be used repeatedly. The Isolation Filter is single-use only. If there are not enough Isolation Filters, please purchase the ExoIsolator Isolation Filter (EX11), which contains 10 Isolation Filters.
• Be careful to handle the Isolation Filter because it is very thin and fragile.
• The recommended sample volume is 25 ml. Approximately 5-15 minutes is required for filtration of 25 ml sample.
• When the sample volume is more than 25 ml, the time required for filtration will be longer.
• Filtration of exosome-rich supernatant may cause filter clogging. Divide the supernatant and serve them for filtration individually.

1. Preparation

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2.Setting Isolation Filter onto the Filter Holder

 

 

Notice

Make sure there are no wrinkles on the surface of the filter. If any wrinkles appear, gently smooth them out by hand or using the provided tweezers.

 

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3. Purificaition of exosomes

 

 

 

• User Guide Video is available on our website.​
• To increase the concentration of the exosomes, decrease the volume of PBS used in step 12-13. Be aware that reducing PBS volume may result in lower recovery of exosomes.
• Make sure all surface of the Isolation Filter being flushed to collect the exosomes in high recovery rate.

Isolation of exosomes from HEK293S cell supernatant using ExoIsolator

1. HEK293S cells suspended in 30 ml serum free medium at 5×10⁵ cells/ml were placed in a 125 ml baffled Erlenmeyer flask. The cells were shaken at 155 rpm at 37°C for 2 days in a 5% CO₂ incubator.
2. After centrifuging at 1,500 rpm for 5 min, the cell supernatant was transferred to a 50 ml conical tube and stored at 4℃.
3. The collected cells were resuspended in 30 ml of fresh serum free medium and cultured for another 2 days in the same conditions.
4. After centrifuging at 1,500 rpm for 5 min, the cell supernatant was transferred to a 50 ml conical tube and stored at 4℃.
5. The cell supernatants collected in steps 2–4 were mixed and passed through a 0.22 μm filter to prepare the prefiltered cell supernatant.
6. The Isolation Filter was put on the support screen and the funnel was settled on the Isolation Filter.
7. PBS (4 ml) was poured onto the Isolation Filter and aspirated.
8. After aspiration, 25 ml of pre-filtered cell supernatant was poured onto the Isolation Filter.
9. After aspiration, 2 ml of PBS was poured onto the Isolation Filter and aspirated. This operation was repeated three times.
10. After aspiration, the Isolation Filter was flushed repeatedly with 250 μl PBS using a micropipette and the suspension was collected by pipetting.
11. The PBS containing the exosomes was transferred to a 1.5 ml microtube.
12. Steps 10–11 were repeated.
13. The PBS containing exosomes were collected as 500 μl of total volume and were examined by nanoparticle tracking analysis (NTA) and western blotting.

 

Comparison of exosome purity collected by UC and ExoIsolator

[Purification of exosomes by UC]

1. The cell supernatant from HEK293S cells was passed through a 0.22 μm filter to prepare the pre-filtered cell supernatant.
2. The pre-filtered cell supernatant (25 ml) was transferred to a centrifuge tube and centrifuged at 100,000 × g for 2 hours (himac CP80NX, Hitachi).
3. After decantation, the pellet was then suspended in 6 ml PBS and centrifuged at 100,000 × g for 2 hours.
4. After decantation, the pellet was then suspended in 100 μl PBS.
5. The PBS containing the exosomes was transferred to a 1.5 ml microtube.
6. The centrifuge tube was washed with 100 μl PBS. PBS used for washing was transferred to the 1.5 ml microtube. This step was repeated three times. (total volume:300 µl )
7. The PBS containing the exosomes was centrifuged at 10,000 × g for 5 min. The supernatant was transferred to a new 1.5 ml microtube.

[Nanoparticle Tracking Analysis (NTA)]

Particle number of purified exosomes were measured using Nanosight NS300 (Quantum Design; camera level 13; detect threshold 5).

 

[Western blotting]

1. Purified exosomes were mixed with sample buffer without dithiothreitol and boiled at 95°C for 5 min.
2. Samples were separated by SDS-PAGE (SuperSep Ace, 10-20%, 13well; Wako) and transferred to PVDF membranes (Trans-Blot Turbo Transfer System Transfer Pack; Bio-Rad).
3. Following antibodies were used for detection of CD9, CD63, and CD81.

   	Primary antibody	Secondary antibody
   CD9	Monoclonal rabbit anti-CD9 antibody (diluted 1:1000; cat. no. ab236630; abcam)	Polyclonal goat anti-rabbit antibody conjugated with HRP (diluted 1:2000; cat. no. ab97051; abcam)
   CD63	Monoclonal mouse anti-CD63 antibody (diluted 1:1000; cat. no. MEX002-3; MBL)	Polyclonal goat anti-mouse antibody conjugated with HRP (diluted 1:2000; cat. no. ab205719; abcam)
   CD81	Monoclonal mouse anti-CD81 antibody (diluted 1:500; cat. no. MEX003-3; MBL)	Polyclonal goat anti-mouse antibody conjugated with HRP (diluted 1:2000; cat. no. ab205719; abcam)

4. The membranes were treated with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific).
5. The protein bands were detected using a Syngene Pxi (Syngene).

1. Kalluri, R.; LeBleu, V. S. The biology, function, and biomedical applications of exosomes. Science. 2020, 367(6478).