Extracellular Vesicle Core

About Us

The CHLA Extracellular Vesicle Core provides the research community with expertise, optimized tools and emerging technologies to support research in the developing fields of ExtraCellular Vesicle and nanoparticles research. The core provides isolation, characterization, analysis and engineering of extracellular vesicles and other nano-sized particles (e.g. viruses, liposomes, nanobubbles). This is the first core of this kind in the country.

Services

The CHLA Extracellular Vesicle (ECV) Core provides the research community with expertise, optimized tools and emerging technologies to support research in the developing fields of ExtraCellular Vesicle and nanoparticles research.

This Core participates in the CHLA Core Pilot Program. To learn more click here.

CHLA welcomes external users to utilize our Core facilities. Please contact Paolo Neviani, PhD at pneviani@chla.usc.edu or call 323-361-8564.

Equipment and Services

The ECV Core provides isolation, characterization, analysis and engineering of extracellular vesicles (i.e. exosomes, 50-150 nm in diameter) and other nanoparticles. The ECV Core will also assist in the generation and analysis of other nanoparticles.

  • Fee Structure
  • Equipment and Software
  • Exo-Flow (exosome flow cytometry)
  • Core Acknowledgment in Publications

Extracellular Vesicle (ECV) Core Fee Structure

Principal Investigators based at The Saban Research Institute

Service/Instrument

Rate

Nanosight Tracking Analysis

Service/Training:$125/hr
Instrument use (trained users): $50/hr
Tech Director time: $75/hr

Isolation of exosomes from cell culture and other biofluids by size exclusion chromatography

[Q:1-9] $50/sample
[Q:10+] $30/sample

Immunoblotting (includes 4 validated primary antibodies and reagents)

[Q:1-17] $280/run

Preparation of vesicle-free FBS

$75 for 50ml

Exo-Flow

$50/sample

Exosomal RNA extraction (including QC by nanodrop)

$15/sample

mRNA or microRNA expression by TaqMan(R) Real-Time qPCR (including reverse-transcriptase and relative quantification, consumables and PCR reagents; users must supply Taqman primer/probes, inquire with core for discounted prices)

$2/replicate

Bio-Rad CFX96 Real-Time system; machine use only

$15/hour

Access for Non-Hospital Researchers

Access to ECV Core facilities for researchers at USC and other institutions can be arranged. Interested researchers should contact the Technical Director to inquire about availability and rates.

NanoSight NS300.

Image result for 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 nanoparticels 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).

 

Bio-Rad fraction collectors (Model 2110) and chromatography columns.

Chromatography columns packed with Sephacryl S-300 high resolution size exclusion chromatography resin are used to isolate exosomes (50-150 nm in size) from body fluids or from the supernatant of cell cultures. After the elution of several fractions by gravity flow, the presence of EVs is be confirmed by protein determination or Nanoparticle Tracking analysis (see above).

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:

  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.

Image result for exo-flow

For the selective capture a magnetic sorting of exosome subpopulations using specific biotinylated antibodies.

Other customer- and project-based services:

  • Isolation and analysis of exosomal RNA.
  • Generation of fluorescent exosomes.
  • Generation of exosomes carrying fluorescent RNA probes.
  • Generation of nanoparticles labeled with specific antibodies.

How to cite the Core in publications:

This work was performed with the support of the ExtraCellular Vesicle Core at The Saban Research Institute of Children's Hospital Los Angeles.