Exosome Research Products

Exosome CD9Exosomes are extracellular vesicles secreted from many cells and are found in various body fluids including serum, plasma, saliva and urine.  Exosomes consist of lipid bilayer membranes, from 40nm to 100nm in diameter, and contain several proteins and nucleic acid such as microRNA, mRNA and DNA.  In recent years, it has been suggested that exosomes may be biomarkers for cancer, diabetes, neurological diseases and various diseases.  Exosomes bear specific protein markers of the endosomal pathway, such as tetraspanins (CD63, CD9 and CD81), heat shock proteins (HSP70) and EpCam depending on their cellular origin.

MBLI offers kits, antibodies, tools, and protocols for the isolation of exosomes as well as for characterization.

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Learn more about the most popular Exosome Research Products

 ExoCap™ Streptavidin Kit

ExoCap™ kits are designed for exosome isolation and enrichment and are optimized for use with serum, plasma and cell culture supernatants. Exosomes are extracellular vesicles secreted by most cell types that contain marker proteins and microRNAs. ExoCap™ Streptavidin Kit is designed for customized isolation and analysis of exosomes or microvesicles, called Extracellular Vesicles (EVs), using the researcher’s biotinylated molecules such as antibodies against exosome surface marker proteins. ExoCap™ uses functionalized Magnosphere™, magnetic micro-particles coated with JSR Life Sciences proprietary hydrophilic polymer to decrease non-specific binding. 

 

Case Study: A Comparison of Exosome Isolation from Blood Collection with Conventional EDTA and TransFix/EDTA Vacuum Blood Collection Tubes

Summary

The exosome profile (CD9, CD63, and CD81 tetraspanin proteins) of normal serum with and without the use of TransFix containing tubes, after storage at 4°C for 4 days was evaluated. Western blot analysis showed detection of all three exosome tetraspanin proteins. NanoSight analysis showed that the TransFix tubes were more likely to capture particles of the correct size range for exosomes and that the particle concentration is higher for the EDTA samples than the TransFix samples. This higher concentration in the EDTA plasma sample is believed to be due to the release of exosomes from platelets during processing and storage at 4°C.

  1. Western blot analysis showed detection of all three exosome tetraspanin proteins.
  2. NanoSight analysis showed that the TransFix tubes were more likely to capture particles of the correct size range for exosomes (1).
  3. The particle concentration is higher for the EDTA samples than the TransFix samples. This higher concentration in the EDTA plasma sample is believed to be due to the release of exosomes from platelets during processing and storage at 4°C (2)(3).

The addition of TransFix at the point of blood collection may reduce the negative impact of platelet exosome release, providing a more accurate picture of the exosome content of samples.

Method

On day 0 a blood draw from donors was performed. Each donor provided 3 x 9 ml of blood drawn into conventional EDTA Vacuum Blood Collection Tubes and 3 x 9 ml of blood drawn into the TransFix/EDTA vacuum blood collection tubes, (Product Code TVT-09-50) from a single venipuncture. The tubes were centrifuged at 300 x g for 20 minutes at room temperature. The upper plasma layer was separated and divided into 1 ml aliquots and samples were stored at 4°C. On day 4, exosomes were isolated from the EDTA and TransFix plasma samples (using an ExoCap™ Streptavidin Kit, MBL International). The isolated exosomes were then analysed by western blot to visualize exosome proteins and by NanoSight to determine the exosome size and concentration.

Results

exosome1

Figure 1:

NanoSight analysis of the conventional EDTA and TransFix plasma samples shows capture of particles expressing the three tetraspanin proteins. The average particle size is higher for the EDTA samples compared to the TransFix samples, which falls in the exosome size range (1). The larger sized particles captured from the EDTA plasma may be a result of protein or exosome aggregation after storage at 4°C for four days.

 

exosome1

Figure 2:

NanoSight analysis of the conventional EDTA and TransFix plasma samples shows capture of particles expressing the three tetraspanin proteins. The particle concentration is higher for the EDTA samples than the TransFix samples. This higher concentration in the EDTA plasma sample is believed to be due to the release of exosomes from platelets during processing and storage at 4°C (2,3).

References

  1. Graça Raposo, Willem Stoorvogel (2013). Review: Extracellular vesicles: Exosomes, microvesicles, and friends [online] 200 (4): 373. Available at: http://jcb.rupress.org/content/200/4/373#ref-104
  2. Heijnen, F.G. et al (1999). Activated Platelets Release Two Types of Membrane Vesicles: Microvesicles by Surface Shedding and Exosomes Derived From Exocytosis of Multivesicular Bodies and Granules. Blood. Volume 94, p 3791-3799. Available at http://www.bloodjournal.org/content/94/11/3791?sso-checked=true
  3. Aatonen, A.T et al (2014). Journal of Extracellular Vesicles. Volume 3, 2014 - Issue 1. Available at https://www.tandfonline.com/doi/full/10.3402/jev.v3.24692

 

 

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