• Novel affinity method

  • Isolating by a PS-binding molecule^※
  • Low background
  • Mild elution by a chelating reagent under a neutral pH condition

  ※High purity exosomes can be easily isolated by PS affinity method.

  

  High purity and intact exosomes

• No required ultracentrifugation

  • Easy operation using magnetic beads
  • Optimized protocol
  
  

  

  High reproducibility

 

Target samples: cell culture supernatant, serum, plasma, urine, etc.

　

Preprocessing protocols for various samples

This is the section to prepare samples. When exosomes and other large EVs (microvesicles) are needed, prepare 1,200 x g supernatant ^{※ 1} as a sample.

Additionally, when highly purified exosomes are needed, prepare 10,000 x g supernatant ^{※ 2} as a sample. This protocol for sample preparation is set for cell culture medium, serum, and plasma. When other body fluids are used, please examine the appropriate preprocessing protocol by referring to the protocol for serum and plasma.

※1 When exosomes and large EVs are needed, use 1,200 x g sup. franction as sample.
※2 When exosomes are needed, use 10,000 x g sup. fraction as sample.
※3 When Large EVs are needed, use ppt of Large EVs obtained by centrifugation at 10,000 x g as sample after suspending it with TBS.
※4 The concentration step is an option when using a large volume (～ 50 mL) of cell culture supernatant as a sample for purification. However, since recovery efficiency improves, please perform it as much as possible.
※5 Because EV-Save™ contains polymers, its use is not recommended for preparation of proteomic analysis samples.

Note : a volume of large or small sample

In the case of large volume

The concentration of the sample is recommended when using a large volume (~50 mL) of cell culture supernatant as a sample for purification. Since recovery efficiency improves, please perform it as much as possible.

In the case of small volume

Add the appropriate volume of TBS into samples to reach the volume of 0.5 mL to obtain the better mixture of the Exosome Capture-immobilized beads with the sample. (Example: 100-200 μL → 500 μL)

 

Reference data : NTA of exosomes and large EVs

1 mL of K562 cell culture supernatant (collected after stimulating exosome secretion with monensin sodium salt) was centrifuged at 10,000 x g to isolate supernatant and precipitate (suspended with 1 mL of TBS) fractions.

Then, extracellular vesicles were purified from both fractions with MagCapture™ Exosome Isolation Kit PS and analyzed using NanoSight LM10.

　

Outline of Procedure of MagCaptureTM Exosome Isolation Kit PS

Analysis of extracellular vesicles purified from serum / plasma / urine samples

Comparing the yield of exosomes isolated from human normal serum

Exosomes were isolated from normal human serum by using MagCapture™, ultracentrifugation and affinity method with antibody against surface antigen of exosome, followed by Western Blot with the anti-CD9 and anti-CD63 antibodies.

Lane 1: Ultracentrifugation  Lane 2: MagCapture™ Lane 3: Exosome Isolation reagent with antibody against CD9 [Supplier A]  Lane 4: Exosome Isolation reagent with antibody against CD63 [Supplier A]  Lane 5: Exosome Isolation reagent with antibody against CD81 [Supplier A]  Lane 6: Exosome Isolation reagent with antibodies [Supplier A]  (antibody beads-mixture of CD9, CD63, CD81 & EpCAM)

Detection antibody Anti-CD9 rabbit pAb, System Bioscience Anti-CD63 rabbit pAb, System Bioscience  About data of WB Each signal corresponds to 150μL of the serum sample. 15μL of eluate and 5μL of 4×SDS sample buffer were mixed and applied all in each well.

 

Isolating exosomes from human EDTA-plasma

Exosomes were isolated from EDTA-plasma, EDTA-plasma (buffer-exchanged), and serum by using MagCapture™, followed by Western Blot with the anti-Flotillin-2 antibody.

Lane 1: EDTA-plasma Lane 2: EDTA-plasma (buffer-exchanged)  Lane 3: Serum

Detection antibody Anti-Flotillin-2 mouse mAb (29/Flotillin-2), BD Bioscience Sample volume 1 mL each About data of WB Each signal corresponds to 150μL of various samples. 15μL of eluate and 5μL of 4×SDS sample buffer were mixed and applied all in each well.

 

Comparing the yield of exosomes isolated from human normal urine

Exosomes were isolated from normal human urine by using MagCapture™, ultracentrifugation and polymerbased precipitation, followed by Western Blot with the anti-CD9 antibody.

Comparison of recovery amount of marker protein

Lane 1: Polymer-based precipitation Lane 2: Ultracentrifugation Lane 3:Mag Capture™

Detection antibody Anti-CD9 rabbit pAb, System Bioscience Sample volume 1 mL each About data of WB Each signal corresponds to 150μL of the urine sample. 15μL of eluate and 5μL of 4×SDS sample buffer were mixed and applied all in each well.

 

Comparison of NTA

Size Distribution Mean: 176 +/- 1.6 nm Mode: 117 +/- 4.5 nm SD: 96 +/- 5.8 nm		Size Distribution Mean: 191 +/- 9.4 nm Mode: 113 +/- 2.8 nm SD: 88 +/- 7.4 nm		Size Distribution Mean: 107 +/- 1.2 nm Mode: 86 +/- 1.2 nm SD: 42 +/- 1.0 nm

Analysis of extracellular vesicles purified from culture supernatant samples

The yield and purity were compared for exosomes isolated from K562 (human chronic mylogenous leukemia: CML) cell culture supernatants (serum-free medium, or 10% Exosome-depleted FBS medium) by using MagCapture™, ultracentrifugation and polymer-based precipitation method.

MagCapture™ Exosome Isolation Kit PS

Exosomes were recovered from 1 mL of pretreated (10,000×g, 30 min.) cell culture supernatant of K562 cells (serum-free medium or 10% exosome-depleted FBS ※ 1 included medium) by using PS affinity method's standard protocol (reaction time: 3 hours)

Ultracentrifugation

10 mL of pretreated (10,000×g, 30 min.) cell culture supernatant of K562 cells (serum-free medium or 10% exosome-depleted FBS ^{※ 1} included medium) were ultracentrifuged at 110,000×g, 70 min., and the precipitates were suspended with TBS. Then, the suspension was ultracentrifuged again and the precipitated pellet was recovered as exosome sample.

Polymer-based precipitation

Exosomes were collected from 1 mL of pretreated (10,000×g, 30 min.) cell culture supernatant of K562 cells (serum-free medium or 10% exosome-depleted FBS^※1 included medium) by Supplier A’s product in accordance with manual (Precipitation time: overnight).

※1: Centrifuged for 2 hours at 110,000 × g, and the supernatant was collected so as not to take up the precipitate.

 

Electron microscopic analysis and Nano analysis of isolated exosomes

The particle size of exosomes from K562 cell culture supernatant (serum-free medium) using MagCapture™, ultracentrifugation and polymer-based precipitation, respectively was determined by using NanoSight LM-10. The collected exosomes (2-4 x 10¹⁰ particles) were fixed by 2% paraformaldehyde and analyzed by electron microscopy.

Electron microscope images were provided by Dr. R. Hanayama at Graduate School of Medicine, Kanazawa University and Dr. Nakai at iFReC, Osaka University.

Comparing the recovery amount and purity of exosomes (serum-free)

Exosomes were collected from cell culture supernatant of K562 cells (serum-free medium) by MagCapture™, ultracentrifugation and polymer-based precipitation. The recovery amount and purity were analyzed by silver staining and Western Blot with anti-CD63, anti-Flotillin-2, and anti-Lamp-1 antibodies.

Comparing the recovery amount and purity of exosomes (10% exosome-depleted FBS)

The exosomes were collected from cell culture supernatant of K562 cells (10% exosome-depleted FBS included medium) by MagCapture™, ultracentrifugation and polymer-based precipitation. The recovery amount and purity were analyzed by silver staining and Western Blot with anti-CD63, anti-Lamp-1, and anti-Flotillin-2 antibodies. Furthermore, collected samples by each method were analyzed by mass spectrometry and compared the percentage of human-derived peptides from K562 cells.

MASS analysis data was provided by Dr. R. Hanayama at Graduate School of Medicine, Kanazawa University and Dr. W. Nakai at iFReC Osaka University.

Comparison of microRNA and mRNA recovery amount from exosomes derived from normal human serum

Comparison of microRNA and mRNA recovery amount from exosomes prepared by different techniques

After isolation of exosomes from normal human serum samples by ultracentrifugation and the PS affinity method, RNA was recovered using microRNA Extractor SP Kit (Code No. 295-71701). microRNA (let-7a, miR-16, miR-92a, miR-142-3p) and mRNA (GAPDH, PIK3CB) were determined by quantitative PCR and compared it using Ct values.

Comparison of RNA extraction methods from the PS affinity fractions

After isolation of exosomes from normal human serum samples by the PS affinity method, RNA was recovered using microRNA Extractor SP Kit (Code No. 295-71701) or acid guanidinium thiocyanate-phenolchloroform extraction (AGPC method). microRNA (let-7a, miR-16, miR-92a, miR-142-3p) and mRNA (GAPDH, PIK3CB) were determined by quantitative PCR and compared it using Ct values.

Description and results of experiment

Exosomes were purified from K562 cell culture supernatant containing 10% exosome-free FBS using either the PS affinity method, ultracentrifugation, or polymer precipitation. The obtained exosome samples were separated by 10% polyacrylamide gel electrophoresis and the individual whole protein bands were cut out. After in-gel digestion, proteins were identified by liquid chromatography mass spectrometry (LC-MS). Proteins identified in exosome preparations purified by the 3 different methods (n=3 for each method) were also compared for Pair-wise correlations.

Comparison of the top 10 proteins identified by MASS analysis

White columns: human protein derived from EVs
Gray columns: bovine protein contaminants derived from FBS
Red color: marker proteins of EVs
Sample: K562 cell culture Sup. (10% exosome-depleted FBS included)

	PS Affinity	Ultracentrifugation	Polymer Precipitation
1	Heat shock cognate 71 kDa Protein	DNA-dependent protein kinase catalytic subunit	Complement C3
2	Annexin A6	Transferrin receptor protein 1	Alpha-2-macroglobulin
3	Transferrin receptor protein 1	Serum albumin	Fibronectin
4	V-type proton ATPase catalytic subunit A	ATP-dependent RNA helicase A	Serum albumin
5	Flotiillin-2	Tubulin beta-5 chain	Thrombospondin-1
6	Programmed cell death 6-interacting protein	Heat shock cognate 71 kDa protein	Complement C4
7	4F2 cell-surface antigen heavy chain	Fatty acid synthase	Alpha-1-antiproteinase
8	Annexin A1	4F2 cell-surface antigen heavy chain	Apolipoprotein B-100
9	Kinase D-interacting substrate of 220 kDa	U5 small nuclear ribonucleoprotein helicase	Hemoglobin fetal subunit beta
10	Annexin A2	Tubulin beta-4B chain	Tubulin beta-5 chain

 

Comparison of Pair-wise correlation

Assay of protein concentration in exosomes using Protein Assay BCA Kit

PProtein Assay BCA Kit, capable of assaying total protein concentration in solution using bicinchoninic acid (BCA), is the most widely used protein assay kit. It is based on the principle of reduction of Cu²⁺ to Cu⁺ by protein under basic conditions. Chelate formation between Cu⁺ and BCA generates a purple-colored chelate. Since the purple color becomes more intense in proportion with protein concentration, the protein concentration is determined by comparing this absorbance at 562 nm with a standard curve of absorbance from varying bovine serum albumin (BSA). 
Here, the protein concentration in an exosome preparation purified from cell culture supernatant using MagCapture™ Exosome Isolation Kit PS was determined according to the high-sensitivity protocol for Protein Assay BCA Kit.

Description and results of experiment

An exosome solution purified using MagCapture™ Exosome Isolation Kit PS pipetted into a 96-well plate in 25 μL aliquots and then 200μL of a mixture of Reagent A and Reagent B included in Protein Assay BCA Kit was added to each well. After subsequent incubation at 60℃ for 30 minutes, the absorbance was measured at 560 nm (Figure 1). The result demonstrated that the protein concentration in a purified exosome preparation was able to determine using the high-sensitivity protocol for Protein Assay BCA Kit.

Figure 1. A BSA calibration curve determined using Protein Assay BCA Kit and assay of protein concentration of a purified exosome preparation

 

Overview of BCA assay protocol

Since protein concentrations of exosome preparations are rather low, use undiluted samples for BCA protein assay.

1. Pipette 25μL per well of standard BSA solutions (250, 125, 62.5, 31.25, 15.625μg/mL) and a standard BLANK into a 96-well plate.
2. Pipette 25μL per well of a purified exosome preparation and Elution Buffer (BLANK) on a 96-well plate.
3. Add 200μL per well of a mixture of Reagent A and Reagent B (A: B=50: 1) of Protein Assay BCA Kit (Code: 297-73101) to each sample-containing well.
4. Incubated the plate at 60℃ for 30 minutes.
5. Allow the plate to cool at room temperature.
6. Measure the absorbance at 560 nm.

Experiment overview

Exosomes purified by MagCapture™ Exosome Isolation Kit PS were labeled with PKH67 (Sigma) and confirmed their ability to be incorporated into HeLa cells.

Exosome labeling with PKH67

1. Purify exosomes using MagCapture™ Exosome Isolation Kit PS (from COLO201 cell culture supernatant on the day of experiment). 
   * To prevent loss of labeled samples due to adsorption in Step 6-7 Gel filtration, addition of EV-Save™ Extracellular Vesicle Blocking Reagent to the elution buffer included in the kit is recommended. Using the elution buffer containing EV-Save™, the sample can be prepared with the minimal loss.
2. Determine the protein concentration and particle concentration by BCA Assay and using NanoSight.
3. Dispense the exosome sample solution corresponding to 3 μg protein* in a 1.5 mL tube. 
   * Prepare an appropriate amount as needed for the experiment.
4. Dissolve 2.0 μL PKH67 linker in 0.25 mL of Diluent C (provided with the PKH67 kit) - 4 x Dye solution* 
   * Prepare an appropriate amount as needed for the experiment.
5. Add 1/3 volume of 4 x Dye solution to the exosome sample, mix, and incubate the mixture at room temperature for 5-10 minutes.
6. Equilibrate Exosome Spin Columns (MW 3000) (Thermo #4484449) with PBS according to the protocol provided with the product.
7. Apply 100μL of each sample to each spin column equilibrated as described above* and centrifuge at 750 x for 2 minutes to separate unbound dye from labeled exosomes.
   * Because the maximum loading volume is 100 μL per column, multiple columns corresponding to the sample volume are recommended to be kept on hand where the sample amount is more than 100 μL.
8. Add the solution containing labeled exosomes* to HeLa cells seeded on a dish on a preceding day. After 24 hours, perform microscopic observation and flow cytometry.
   * Adjust the number of exosomes added.

• Results of microscopic observation

  

• Results of flow cytometry

  

Figure 1. Observation of the uptake of PKH-labeled exosomes into HeLa cells. Exosomes were isolated from COLO201 cell conditioned medium using MagCapture™ Exosome Isolation Kit PS (total protein 3 μg, particle number 1 x 10¹⁰), followed by labeling using PKH67 Green Fluorescent Cell Linker Kit (Sigma). The uptake of the labeled exosomes into HeLa cells was confirmed by fluorescence microscopy (left) and flow cytometry (right).