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Isolation
Isolation and Purification

Exosomes have been found in virtually all biofluids, including plasma, cerebrospinal fluid (CSF), saliva, urine, conditioned cell culture media, ascites fluid, and tissue lysates. With a wide variety of methods and commercial kits available there is still no universal method to isolate exosomes due to the very different nature of the source biofluids. At Alpha Nano Tech a team of experienced scientists will work closely with you to develop an optimal protocol for your specific project and research interest. We fully understand the advantages and disadvantages of each method and can help you maximize the yield and quality of your exosomes. The exosomes can be shipped back to the client if requested. 

Methods for Exosome Isolation
  • Ultracentrifugation 

  • Size exclusion chromatography

  • Affinity-based purification

  • Ultrafiltration

  • Tangential flow filtration

  • Polymer-based precipitation of exosomes

  • Membrane affinity-based isolation

Features
  • Development and optimization of a custom protocol designed specifically for your unique project

  • Flexible options for each specific case

  • Downstream full spectrum characterization services

  • Fast turnaround time

NTA

The most suitable and most cited technique for exosome analysis: counts each particle independently to measure size, concentration and size distribution.

  • Direct analysis in solution 

  • Low detection limit and wide concentration range: E+7-E+9 particles/mL

  • Ability to work in the “Scatter” mode to measure all particles and then analyze the same particles in “Fluorescent” mode to part out and assay EVs labeled with membrane dyes or fluorescently labeled antibodies.
     

Our NTA Services: 
  • Both Nanosight and Zetaview instruments are available

  • 405 nm, 488 nm, 520 nm, and 640 nm lasers are available with corresponding long pass cut off filters for optimal excitation and emission of fluorophores

  • EVs purity assesment by mebrane lavelling

  • In house fluorescent labeling of EVs for fNTA analysis for custom biomarker expression analysis

  • Immunophenotyping by multicolor NTA

  • Cross-validation by TEM, DLS, Laser diffraction, TRPS, MRPS

Nanoparticle Tracking Analysis and
Fluorescent Nanoparticle Tracking Analysis

Schematic representation of NTA and fNTA, NTA analysis in scatter and fluorescent modes of liposomes and Exosomes. The measurement is done with the Zetaview NTA instrument. In the fNTA EV membranes were labeled with ExoGlow fluorescent dye (SBI) and the sample is analyzed in both scatter and fluorescent modes. This allows excluding protein aggregates, membrane fragments and other background particles from the analysis. 

exosome 3.png

Multicolor Fluorescent NTA

While NTA technique is most commonly used to profile EVs and nanoparticles size, size distribution, and concentration, the fNTA enables a whole new field of research applications:

- fluorescent membrane labeling allows to assess EVs samples purity and sort out membranous vesicles out of the total number of particles in solution

-      biomarkers profiling via fluorescently labeled antibodies is a powerful tool to measure up to 4 different biomarker expression on the same sample, which saves you time and precious material

-      RNA/DNA labeling for a perspective class of novel nano-formulations inspired by Moderna’s mRNA vaccine

-      drug loading efficiency to validate the number of cargo-carrying particles

-      drug release kinetics of the fluorescently labeled drug out of nanoparticles

The fNTA technique combines the abilities of NTA, flow cytometry, and fluorescent microscopy with the advantage of nano-scale real-time analysis.

exosome 3.png

Transition Electron Microscopy (TEM) allows imaging of EVs and assessment of size and size distribution. Alpha Nano Tech provides statistically relevant data based on the imaging and analysis of thousands of particles per sample.

Immunogold TEM is a direct and powerful tool for the phenotypic characterization of EVs in addition to traditional TEM and protein analysis.

 

TEM is the best method to cross-validate particle size and size distribution data acquired from other methods, such as Nanoparticle Tracking Analysis (NTA), dynamic light scattering (DLS), or tunable resistive pulse sensing (TRPS). 

Cryo-electron microscopy (Cryo-TEM or Cryo-EM by Talos Arctica, Thermo) is another powerful tool to study nanoparticles, including exosomes. The samples are frozen in liquid propane-ethane mixture and thus the shape and micro-structure are preserved better compared to the traditional TEM method.

Our experienced scientists are always available to provide guidance in sample preparation, experimental design, and data analysis. 

 

  • unaltered by drying unlike conventional TEM

  • fast specimen freezing coverts water into transparent vitreous ice

  • ice preserves biomolecules in a rigid state allowing high resolution imaging

  • sub 10 Å resolution (1 nm) allows imaging near-atomic structures

  • quick-fire imaging allows using high power beam up to 200 kV