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
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Ultracentrifugation
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Size exclusion chromatography
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Affinity-based purification
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Ultrafiltration
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Tangential flow filtration
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Polymer-based precipitation of exosomes
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Membrane affinity-based isolation
Features
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Development and optimization of a custom protocol designed specifically for your unique project
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Flexible options for each specific case
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Downstream full spectrum characterization services
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Fast turnaround time
The most suitable and most cited technique for exosome analysis: counts each particle independently to measure size, concentration and size distribution.
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Direct analysis in solution
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Low detection limit and wide concentration range: E+7-E+9 particles/mL
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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:
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Both Nanosight and Zetaview instruments are available
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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
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EVs purity assesment by mebrane lavelling
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In house fluorescent labeling of EVs for fNTA analysis for custom biomarker expression analysis
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Immunophenotyping by multicolor NTA
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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.
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.
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.
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unaltered by drying unlike conventional TEM
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fast specimen freezing coverts water into transparent vitreous ice
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ice preserves biomolecules in a rigid state allowing high resolution imaging
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sub 10 Å resolution (1 nm) allows imaging near-atomic structures
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quick-fire imaging allows using high power beam up to 200 kV
Our experienced scientists are always available to provide guidance in sample preparation, experimental design, and data analysis.
Our EM exosome services:
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Negative staining
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Immunoelectron microscopy
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Size distribution analysis
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Cryo-electron microscopy
Features
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Qualitative and quantitative analysis
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Highly experienced scientific team
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Consistent reproducible results
Electron microscopy
Microfluidic Resistive Pulse Sensing (MRPS)
by nCS1, Spectradyne
Microfluidic Resistive Pulse Sensing (MRPS) uses electrical sensing to measure the diameter of each particle as it passes through a nanoconstriction, providing real-time sizing and concentration information.
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Resistive pulse sensing and size measurements of individual nanoparticles in fluid
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Particle size distribution with absolute concentration
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Particle size analysis range: 50 nm to 10 μm in diameter
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Concentration range: approx. 1E+6 — 1E+11 particles/mL
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Max particle detection rate: approx. 10,000 particles/s
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Nanoparticles of any material type (transparent & opaque, conducting & insulating)
Resistive pulse sensing
A single nanoparticle increases the electrical resistance of the suspending fluid as it passes through the nanoconstriction, giving rise to a brief voltage pulse. The size of the pulse is proportional to the nanoparticle volume; the amplitude and duration of the pulse gives the particle diameter and the mean velocity, a method known as resistive pulse sensing. Longer measurements accumulate many pulses.
Concentration measurements
Software-based analysis is used to combine measurements of a large number of particles into a histogram of particle diameters, with the vertical scale in units of nanoparticle concentration. After removing the dependence on the width of histogram binning, the resulting Concentration Spectral Density (CSD) has units of nanoparticles per unit volume per unit diameter (e.g., particles · mL-1 · nm-1). Because of the fast measurement capability of the nCS1, good statistics can be obtained in a few seconds.
Exosome RNA Analysis
Exosomal miRNAs are identified as characteristic biomarkers for different types of cancer, including lung, colorectal, ovarian, etc. Exosomal miRNA-based test has been recently approved by the FDA for diagnostics of prostate cancer in patients with ambiguous PSA test results. At Alpha Nano Tech it is our goal to provide tools and expertise to accelerate your exosomal RNA research.
Our RNA Profiling Services:
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RNA isolation
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Total RNA content
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RNA QA/QC
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Next Generation Sequencing
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Bioinformatics analysis
Features
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Sequence ultra low inputs from small sample quantities
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Consultation and data analysis advise throughout the process
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RNA QC on every step
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Fast turnaround time
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Raw data archive download
Biomarkers Expression and Protein Analysis
EVs must carry a panel of proteins to classify as exosomes. Alpha Nano Tech provides complete biomarker profiling as an essential step of EV isolates characterization, ensuring at least three exosome relevant biomarker present and negative controls absent in the sample as advised by the International Society of Extracellular Vesicles.
At Alpha Nano Tech we approach EV biomarker profiling with a variety of techniques to help you accurately characterize samples of any complexity.
Our Protein Profiling Services:
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Total protein content - UV rapid test or BCA prtotein content analysis
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fNTA immunophenotyping - quantifying biomarkers expression via fluorescent antibodies labeling and detection
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Westen Blot analysis - capillary Western Blot protein analysis via Jess (Protein Simple)
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LC-MS/MS analysis for in-depth assessment of protein content by orbitrap Exactive Q (Thermo)
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ELISA - quantitative analysis of custom biomarker targets
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Flow cytometry biomarker assessment
Lipidomics
Alpha Nano Tech LLC is excited to offer the latest in advanced lipidomic analysis using Thermo Q Exactive. Lipidomic analysis allows us to characterize the complex lipid content of extracellular vesicles (EVs) in greater detail than ever before. By understanding the lipid content of your EVs, you can gain a deeper understanding of their biogenesis and function.
Our lipidomic analysis provides a comprehensive evaluation of the lipid composition of your EVs, allowing you to identify not only the different lipid classes present, but also the specific lipid species within those classes. This information can provide valuable insights into the cellular and molecular mechanisms underlying the biogenesis of EVs, as well as their physiological and pathological roles.
Furthermore, understanding the lipid content of your EVs can help in the development of EV-based therapeutics. Lipids play a crucial role in the stability, targeting, and delivery of EVs, making it essential to have a comprehensive lipidomic analysis of your EVs to ensure optimal efficacy.
Contact us today to learn how we can help you better understand the lipid content of your EVs and take your research to the next level.
Flow Cytometry
Flow Cytometry is a well-established technique for measurement of cell characteristics, which can include cell size, cell count, cell cycle and more. This technique allows researchers to get highly specific information about individual cells. Typical samples include everything from a cell line expressing Green Fluorescent Protein to heterogeneous cell populations harvested from tissues. The most critical requirement for efficient and effective flow cytometry analysis is that the sample be a single-cell suspension. This helps ensure that every cell is analyzed independently.
Recently with advancement in sensitivity and resolution of Flow Cytometry, technique was adopted towards analysis of EVs for identification and characterization. This allows for the rapid quantification, biochemical profiling, and functional analysis of exosomes, thus providing valuable insights into their roles in intercellular communication, disease progression, and therapeutic applications.
Flow Cytometry enables the detection of exosomes based on their size, surface markers, and intrinsic properties, making it an excellent tool for exosome heterogeneity investigations.
Get in touch with us today to learn more about our expert Flow Cytometry services.
Alpha Nano Tech LLC provides services using:
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CytoFlex by Beckman Coulter
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MacsQuant by Miltenyi