Extracellular Vesicle Core

NanoSight NS300

The Malvern Panalytical NanoSight NS300 uses the technology of Nanoparticle Tracking Analysis (NTA). This unique technology utilizes the properties of both light scattering and Brownian motion in order to obtain the size distribution and concentration measurement of particles in liquid suspension. A laser beam is passed through the sample chamber, and the particles in suspension in the path of this beam scatter light in such a manner that they can easily be visualized via 20x magnification microscope onto which is mounted a camera. The camera operates at 30 frames per second (fps), capturing a video file of the particles moving under Brownian motion. The software tracks many particles individually and using the Stokes-Einstein equation calculates their hydrodynamic diameters.

• Automated analysis of the size distribution and concentration of all types of nanoparticles and extracellular vesicles from 20nm to 1000nm in diameter.
• Equipped with a 532 nm green laser and a 565LP fluorescence filter for the detection of fluorescently labeled vesicles (e.g. CellMask Orange, PKH26, Dil dyes).

ÄKTA™ start

ÄKTA™ start is a laboratory scale preparative chromatography system designed as a stand-alone system, with intuitive design, simple flow path, and user-friendly interface.

ÄKTA start offers the following features:

• Compact solution for quick and reproducible purifications
• Sample injection and fraction collection options
• Quick start methods and templates for common purification techniques
• Software enabled real-time monitoring

The Core utilizes the ÄKTA start system for Extracellular Vesicle Isolation by Size Exclusion Chromatography.

EV core users may request access to the instrument and will be able to independently perform a variety of applications, including but not limited to:

• Desalting or buffer exchange
• Affinity Chromatography
• Ion exchange chromatography
• Size exclusion chromatography

ExoView R100

The ExoView instrument is a step forward in characterization capabilities within the extracellular vesicle field. The fully automated platform, based on Single Particle Interferometric Reflectance Imaging Sensor (SP- IRIS) technology, provides multi-level and comprehensive measurements for exosome particle size analysis, exosome count, exosome phenotype, and biomarker colocalization. The ExoView platform provides previously unattainable information in a single and bias free sample workflow.

ExoView works without the need for sample purification and brings the researcher one step closer to analyzing a sample in its natural state. ExoView is an affinity-based technology that allows specific populations of exosomes and other extracellular vesicles to bind in a multiplexed manner to a functional ExoView® chip. The fully automated instrument can measure 9 samples automatically, direct from sample, saving cost, time, and reducing purification biases.

Advantages:

1. Biomarker colocalization:
   • Quantify relative protein expression of up to 4 proteins per exosome
2. Count extracellular vesicles:
   • Count the number of antigen-positive exosomes directly from sample, no purification required
3. Extracellular vesicle size:
   • Acquire high-resolution measurements of the size of individual antigen-positive exosomes
4. Extracellular vesicle cargo:
   • Probe for exosomal luminal proteins and cargo
5. Fluorescence:
   • Detect lowest abundance proteins with single binding event sensitivity across three color channels
6. Streamlined workflow:
   • Automated analysis of up to 9 samples with single button sample analysis
7. Purification not required:
   • Detect the changes in your sample, not the biases from your purification technique
8. Multiplexed sample analysis:
   • Characterize multiple populations of exosomes from a single sample using up to 6 surface markers

For more information: www.nanoviewbio.com

Beckman Coulter Optima XE-90K

The Beckman Optima™ XE-90K Preparative Ultracentrifuge is suitable for separation of small particles such as small extracellular vesicles, viruses, viral particles, proteins and/or protein complexes, lipoproteins, RNA, and plasmid DNA.

Several rotors (swing-out and fixed angle) for different applications are available. Please inquire with core for user training.

Characterization of Exosomes by Immunoblotting with Bio-Rad Protean and Trans-Blot System

Exosome-containing samples are characterized by immunoblotting with previously validated antibodies.

Four markers are analyzed according to the International Society of Extracellular Vesicles https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4275645/:

1. Transmembrane or lipid-bound extracellular proteins (e.g. CD9, CD63 or CD81); Argues presence of a membrane in the isolate; present or enriched in EVs/exosomes.
2. Cytosolic proteins (e.g. TSG101, Alix); With membrane- or receptor-binding capacity; present or enriched in EVs/exosomes.
3. Intracellular proteins (e.g Calnexin); associated with compartments other than plasma membrane or endosomes; absent or under-represented in EVs/exosomes.
4. Extracellular proteins (e.g. Fibronectin-1); binding specifically or non-specifically to membranes, co-isolating with EVs; variable association with EVs.
5. Apolipoproteins (e.g Apo-B100); for complex biofluids such as plasma or serum the presence of low-density lipoproteins (VLDLs, LDLs) is an indication of purity of the EV preparation.

Tangential Flow Filtration – KrosFlo TFF by SpectrumLabs

Tangential flow filtration (TFF) is a rapid and efficient method for separation and purification of biomolecules. See how it works here.

It can be applied to a wide range of biological fields such as immunology, protein chemistry, molecular biology, biochemistry, and microbiology. TFF can be used to concentrate and desalt sample solutions ranging in volume from a few milliliters to several liters. It can be used to fractionate large from small biomolecules, harvest cell suspensions, and clarify fermentation broths and cell lysates.

For example, TFF can be used to concentrate large volumes of EV-containing samples (e.g. conditioned medium) to a more manageable volume prior to EV isolation. Please inquire with the core for user training.

1. Considerations on how to pick the best isolation methods.

2. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. Thery et al, JEV. 2018 Nov 23;7(1):1535750. doi: 10.1080/20013078.2018.1535750 (https://www.tandfonline.com/doi/full/10.1080/20013078.2018.1535750)

How to cite the Core in publications:

The EV work was performed in the Extracellular Vesicle Core at The Saban Research Institute, Children's Hospital Los Angeles. 

Publications made possible by Core support:

• Xin Tang, Cheng Chang, Jiacong Guo, Vadim Lincoln, Mei Chen, David T. Woodley, and Wei Li. Department of Dermatology and the Norris Comprehensive Cancer Centre, University of Southern California Keck Medical Centre. Tumour-Secreted Hsp90a on the External Surface of Exosomes Mediates Tumour-Stromal Cell Communication via Autocrine and Paracrine Mechanisms. Sci Rep. 2019 Oct 22;9(1):15108
  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6805946/
   
• Paolo Neviani, Petra M Wise, Mariam Murtadha, Cathy W Liu, Chun-hua Wu, Ambrose Y Jong, Robert CSeeger and Muller Fabbri Natural Killer-derived exosomal miR-186 inhibits neuroblastoma growth and immune escape mechanisms. Cancer Res December 12 2018 DOI: 10.1158/0008-5472.CAN-18-0779
  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6428417/