Product catalog and Reference Materials
|Exosome Research Products Vol. 1||Poster Presentation in ISEV 2017||Exosome Video Vol. 1:
Outline of Procedure of MagCapture™ Exosome Isolation Kit PS
In recent years, research of extracellular vesicles (EVs)
has been advancing at an accelerating pace. While the
number of scientific articles on EVs published in 2011 was
approximately two hundreds, the number increased to
more than one thousand in 2016 and involvement of EVs
in various physiological functions and pathogenic mechanisms
has been suggested. Although EVs are roughly classified into at least two categories: exosomes derived from
endosomes and microvesicles derived from plasma membrane,
it is difficult to strictly separated them from each
other by differential centrifugation, the technique most
frequently used for purification of EVs at present, and the
EVs not sedimenting at 10,000×g are called "small EVs"
(mainly composed of exosomes) for convenience.1)
Exosomes are small membrane vesicles (approximately 30-100 nm in diameter) secreted by various cells and present in most body fluids (e.g., blood, urine, and spinal fluid) and cell culture liquids. Exosomes, membrane vesicles surrounded by a lipid bilayer, are generated within intracellular vesicles called "multi-vesicular endosomes" and released into the extracellular space by fusion of multi-vesicular endosomes with the cell membrane. Exosomes contain proteins from secretory cells, including those of endosome origin (e.g., ESCRTs), those involved in intracellular transport (e.g., Rab GTPase), and those of cell membrane origin (e.g., CD63 and CD81), as well as RNAs. Exosomes also contain the cell membrane of secretory cells and lipids from the endosome membrane (cholesterol and sphingomyelin, etc.).2)
Although exosomes had long been considered to be involved in release of unnecessary cell contents, exosomes are recently attracting attentions of researchers as new mediators of cell-cell communication transporting biomolecules such as lipids, proteins, and RNAs in vivo. In addition to clarifi cation of physiological or pathophysiological functions of exosomes, research aiming at clinical application of these functions is rapidly in progress, particularly focusing on diagnostic and therapeutic application as well as development of biomarkers.
1) Kowal J, et al. (2016) Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proc Natl Acad Sci U S A, 113(8): E968-977.
2) Colombo M, Raposo G, & Thery C (2014) Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol, 30: 255-289.
- 1. LINKS
- 2. PS affinity method
- 3. Application of the PS affinity method to ELISA
- 4. Related Products
- 5. Exosome Related Products Q&A
- Exosome Research Products Vol. 1 (9,232KB/28p)
- Poster Presentation in ISEV 2017 (303KB/1p)
- Exosome Video Vol. 1: Outline of Procedure of MagCapture™ Exosome Isolation Kit PS (65.39MB/MP4)
- PS Capture™ Exosome ELISA Kit (Anti Mouse IgG POD)
- PS Capture™ Exosome Flow Cytometry Kit
- MagCapture™ Exosome Isolation Kit PS
- Anti-CD63, monoclonal antibody (3-13)
2. PS affinity method
Exosomes are membrane vesicles (30-100 nm in diameter) released from various cells and shown to function
as transmitters of encapsulated nucleic acid (mRNA, microRNA) and proteins between remote cells. Their roles
as a communication tool in cell-cell signal transduction and as a potential biomarkers for various diseases
including cancer have recently been attracting research interest.1, 2) Accordingly, exosome research has been
spreading across a wide variety of research areas in recent several years. Nevertheless, experimental techniques
currently available for exosome research are still under development and many issues remain to be improved.
For example, among conventional techniques for exosome purification, ultracentrifugation and polymer precipitation (commercially available kits) have been shown to yield exosome preparations with large amounts of contaminants that seriously interfere with subsequent experiments. On the other hand, antibody-based affinity method and density gradient centrifugation are capable of purifying highly purified exosomes but are incapable of yielding intact exosomes, thus it is difficult to analyze their original physiological functions of exosomes.
Furthermore, Western Blotting and ELISA widely used for exosome detection have several disadvantages including the requirement of relatively large amounts of exosomes and difficulties in the detection of lowexpression marker proteins.
Accordingly, we have developed a new exosome analysis tool to resolve various problems in experimental techniques for exosome research as mentioned above. This new technique is described below. Data on isolation of high-purity extracellular vesicles using MagCaptureTM Exosome Isolation Kit PS are presented on pages 7-13, while data on high-sensitivity detection of extracellular vesicles using PS CaptureTM Exosome ELISA Kit PS are shown on page 16 and thereafter.
The exosome membrane contains proteins and lipids derived from secretory cells. Among those components,
phosphatidylserine (PS) is known to be oriented inside the cell membrane of intact cells by the enzymatic
activity of lipid flippase, and be known to be exposed also on the outer surface of exosome membrane.3) In
addition, T-cell immunoglobulin domain and mucin domain-containing protein 4 (Tim4), the receptor involved in
phagocytosis of apoptotic cells by macrophages, is known to bind PS via the IgV domain located within the
extracellular region in a calcium ion-dependent manner.4)
Based on these findings, we developed a new and unprecedented method for exosome purification using Tim4-immobilized magnetic beads (capable of capturing exosomes in samples such as culture supernatants and serum in the presence of calcium ions and releasing them by elution with a buffer supplemented with a chelating reagent) in collaboration with Professor Rikinari Hanayama (Department of Immunology, Kanazawa University Graduate School of Medical Sciences) and successfully constructed an exosome purification kit based on these magnetic beads.5) This exosome purification kit, MagCaptureTM Exosome Isolation Kit PS, has realized easy purification of intact exosomes with higher purity than that obtained by any conventional methods for exosome purification. It is currently being established as a new exosome purification method replacing ultracentrifugation, the conventional gold standard.
1) Tkach, M. et al. Communication by Extracellular Vesicles: Where We Are and Where We Need to Go. Cell 164, 1226-1232 (2016).
2) Raimondo, F. et al. Advances in membranous vesicle and exosome proteomics improving biological understanding and biomarker discovery. Proteomics 11, 709-720 (2011).
3) Trajkovic, K. et al. Ceramide triggers budding of exosome vesicles into multivesicular endosomes. Science 319 (5867): 1244–7 (2008).
4) Miyanishi, M. et al. Identification of Tim4 as a phosphatidylserine receptor. Nature 450, 435-439 (2007).
5) Nakai, W. et al. A novel affinity-based method for the isolation of highly purified extracellular vesicles. Sci. Rep. 6, 33935 (2016).
|Extracellular vesicles are captured by phosphatidylserine (PS)-binding proteins and metal ions, then captured extracellular vesicles are eluted with a chelating reagent.|
3. Application of the PS affinity method to ELISA
|We developed PS Capture™ Exosome ELISA Kit by applying affinity binding of Tim4 protein with exosomes. This kit is capable of detecting exosomes at a sensitivity higher than that of conventional ELISA methods immobilization of antibodies against exosome surface markers. Exosomes in samples such as culture supernatants and serum are captured by Tim4 protein on adry plate in the presence of calcium ion. The captured exosomes are detected by a primary antibody against an exosome surface marker protein and a labeled secondary antibody. While a mouse anti-CD63 monoclonal antibody is provided with the kit, a user-provided mouse primary antibody against any other exosome surface marker may also be used for exosome detection.|
|The greatest feature of this kit is that it provides exosomes detection with higher sensitivity than that of Western Blot analysis and conventional product for exosome ELISA. First, the detection limit for exosomes in Western Blotting was examined for comparison with this kit (Figures 1a and b). Western Blot analysis of exosomes purified from COLO201 cells (of human colon adenocarcinoma origin) with an anti-CD63 monoclonal antibody detected exosomes in an amount as small as 75 ng on the protein basis. Next, the detection limits of this kit for exosomes purified from K562 cells (of human leukemia origin) and COLO201 cells were determined to be 49.9 pg and 10.9 pg, respectively, demonstrating that this kit had a detection sensitivity more than 1,000 times higher than that of Western Blotting (Figure 1c). Considering that the detection limits of conventional products for exosome ELISA range approximately several ng to several μg (refer to instruction manuals for individual products), the present results demonstrated that this kit utilizing affinity binding of exosomes to Tim4 via PS has a sensitivity more than 100 times higher than those of conventional ELISA methods involving immobilization of an antibody against an exosome surface protein marker.|
|PS ELISA detected the marker proteins with 50-1,000 times higher sensitivity than WB.|
|Fig. 1 Comparing the detection sensitivities of Western Blot and PS ELISA|
4. Related Products
MagCapture™ Exosome Isolation Kit PS adopts a novel affinity purification method using magnetic beads and phosphatidylserine (PS)-binding protein (PS affinity method). This kit can easily isolate high purity exosomes and other EVs from cell culture medium and body fluids at high yield by a normal microcentrifuge. If higher purity exosomes are needed, please use the supernatant obtained by 10,000 × g centrifugation as the sample. This kit enables the isolation of exosomes and other EVs as intact forms because the captured EVs are eluted from magnetic beads with the metal-chelating reagent at neutral pH. The isolated intact exosomes and other EVs can be used for various applications including electron microscopic analysis, nanoparticle tracking analysis, administration of EVs and analysis of molecular constituents such as proteins, lipids, or nucleic acids.
The kit includes reagents for enzyme-linked immunosorbent assay (ELISA) available for a Qualitative Analysis of extracellular vesicles purified from cell culture supernatant or samples from body fluid as well as a Quantitative Analysis of extracellular vesicles in samples of cell culture supernatant. It can detect extracellular vesicles, which have any surface marker protein, with high sensitivity by using a mouse monoclonal antibody against any surface marker proteins of extracellular vesicles as a primary detection antibody and HRP-conjugated anti mouse IgG antibody of the kit as a secondary detection antibody after extracellular vesicles are captured by a plate on which proteins that specifically bind with phosphatidylserine (PS) on the surface of extracellular vesicles are immobilized.
This product is capable of capturing extracellular vesicles on magnetic beads by a new affinity method (PS affinity method) using magnetic beads and Tim4 protein specifically binding phosphatidylserine (PS) and then detecting surface marker proteins at a high sensitivity by flow cytometry. It realizes direct qualitative analysis of surface marker proteins without purification of extracellular vesicles from cell culture supernatant and body fluid specimens (e.g., serum, plasma). This product requires a primary antibody against the surface marker protein of interest plus a fluorescencelabeled secondary antibody, or a fluorescence-labeled primary antibody against the surface marker protein of interest.
This product is mouse monoclonal antibody against CD63 (lysosome-associated membrane glycoprotein 3: LAMP3). CD63 is a membrane protein with four transmembrane domains. It is used as a marker protein of extracellular vesicles and activated platelets.