Exosomes / Extracellular Vesicles (EVs) are an emerging class of biologics that play a crucial role in intercellular communication and hold tremendous promise. A key challenge in the translation of exosomes is the implementation of robust analytical methods for characterization. The size, heterogeneity, and complex composition of exosomes creates challenges for the use of conventional analytical tools and strategies, especially for critical quality attributes such as purity and potency.

RoosterBio has developed an extensive panel of analytical methods to support process development and manufacturing of exosomes, which we offer to our customers as either a stand-alone service or as an integrated component of a broader process development program. Our standard exosome analytics can be ordered individually or in groups to meet a broad range of customer needs regardless of their cell type or production process.

Contact Us For Your Analytical Needs

We Offer Exosome / Extracellular Vesicle Characterization Assays Specially Designed for Starting & Advanced Programs Alike. All Assays Can Also Be Ordered Individually or in Groups.

Assay	Assay Result	Platform
Nanoparticle Tracking Analysis (NTA)	Particle Number, Particle Size	ZetaView, NanoSight, Flow Nanoanalyzer
% Lipid Bound MemGlow	% Positive Particles by Particle Concentration	Flow Nanoanalyzer
Protein Quantitation	Total Protein	Plate Based Assay
Protein Marker Expression	Presence and Expression of Protein Markers	Capillary Western Blot, Flow Nanoanalyzer
Single-Particle Protein Analysis	% Particles Expressing Target, Mean/Median Molecules Per Particle	Flow Nanoanalyzer
Total RNA Quantitation	Total RNA Content	Bioanalyzer
miRNA Identity Analysis	miRNA Identity via qPCR	QuantStudio
dsDNA Quantitation	Total and Internal DNA Content	Plate Based Assay
Albumin Analysis	Albumin Protein per mL	Plate Based Assay
CD73 Bioactivity	CD73-mediated Conversion of AMP to Adenosine	Plate Based Assay
Angiogenesis	Total Length of Endothelial Cell Networks	Microscopy, Quantitation
Zeta Potential	Degree of Electrostatic Repulsion Between Adjacent Particles	ZetaView
EV Integrity	Detection of intact, biologically derived particles	Plate Based Assay
Macrophage Immunomodulation	% Change in Nitric Oxide Level	Plate Based Assay
RNA Sequencing (RNA-seq)	Quantification of micro and small RNA in sample	Next Generation Sequencing

 

Advanced Equipment for Advanced Analytics

Nanoparticle Tracking Analysis (NTA)

Nanoparticle Tracking Analysis is one of the most widely used methods for characterizing extracellular vesicle identity. This assay utilizes light scattering to quantify the size distribution and concentration of particles in a sample which are two key identity components of extracellular vesicle characterization. Results are provided as a concentration in particles per mL and a size distribution graph of extracellular vesicle diameters. This standardized assay is conducted using the ZetaView x30 Quatt.

% Lipid Bound MemGlow

This extracellular vesicle assay uses the NanoFCM Flow NanoAnalyzer to measure the fluorescence of your sample as a percent of particle population that contains lipids. These results will better inform you of what percentage of your particle concentration is made up of extracellular vesicles. Additional assays can utilize either the ZetaView or nanoFCM Flow NanoAnalyzer to assess tetraspanin make-up of the particle population and custom target readouts for your target of interest upon request.

Protein Quantitation

This protein quantitation assay is a widely used identity and content tool to measure total protein content of an extracellular vesicle sample. Quantitation of protein content is a useful metric for process optimization as well as the characterization of a purified sample. Results are provided as total protein per mL and a bicinchoninic acid assay (BCA), microBCA, or Bradford assay will be used depending on customer preference and sample submission.

Protein Marker Expression

This identity assay utilizes capillary western to measure tetraspanin and cytosolic markers. The tetraspanins CD9, CD63, and CD81 and cytosolic markers Alix and TSG101 are widely expressed in different cell types and considered identity markers of extracellular vesicles. This capillary western blot assay provides a sensitive, quantitative readout via image and densiometric conversion for all five markers in sample preparations. Additional assays utilizing the nanoFCM Flow NanoAnalyzer or capillary western to asses your markers of interest are available upon request.

Single-Particle Protein Analysis

This extracellular vesicle assay utilizes the NanoFCM Flow NanoAnalyzer for single-vesicle analysis, enabling both the characterization of vesicle heterogeneity and the quantification of tetraspanin marker expression within a sample population. Antibody labeling, combined with calibrated fluorescence units, is used to estimate the molecular abundance of target proteins on individual particles. Results are reported as the percentage of particles expressing the target tetraspanin, along with the median and mean number of molecules per particle. The assay supports detection of common MSC-EV tetraspanin proteins (CD63, CD81, CD73) in addition to custom protein targets upon request.

Total RNA Quantitation

This is a quantitative assay that measures RNA content in samples containing EVs. The sample is processed to specifically extract RNA followed by quantification using the Bioanalyzer automated electrophoresis system. The reported measurement reflects the total amount of RNA in the sample. Representative electropherograms will be provided. Each sample is measured in duplicate. Negative and positive controls are run in each assay to ensure proper assay performance.

miRNA Identity Analysis

Quantitatively identify specific miRNAs present in your extracellular vesicle samples. This real-time qPCR analysis provides readouts across several functional categories as well as a minimum of two reference readouts. Additional readouts can be customized to your target of interest upon request.

dsDNA Quantitation

This is a quantitative assay that measures DNA content in samples containing EVs. DNA content in the EV sample is measured using a dsDNA fluorescent reagent and measured using a plate reader. Each sample is measured in triplicate. Negative and positive controls are run in each assay to ensure proper assay performance. Reported DNA content will include total DNA (DNA inside and outside of the particles) and internal DNA (DNA encapsulated within particles).

Albumin Analysis

This quantitative ELISA measures human albumin which is a common media component but impurity when found in extracellular vesicle preparations. Step-wise removal of albumin can be an informative analytical readout as well as a purity indicator for “final product” extracellular vesicle samples. The results of this purity assay are reported as albumin protein per mL.

CD73 Bioactivity

CD73 is a canonical identity marker of MSCs and MSC-derived extracellular vesicles that functions to convert extracellular AMP to adenosine. This assay quantifies the enzymatic activity of CD73 which has been proposed as a bioactivity indicator. The results are provided as AMP Consumption per particle.

Angiogenesis

Angiogenesis is the process of generating new blood vessels from existing ones, and is essential in development, growth, and wound healing. This assay interrogates the pro-angiogenic effect of extracellular vesicles or the secretome by evaluating the total length of endothelial cell networks.

Zeta Potential

Zeta (ζ) Potential is a measure of the surface charge of nanoparticles including extracellular vesicles. A higher magnitude of zeta potential correlates with increased electrostatic repulsion, which prevents particle aggregation and is a stability indicator of colloidal dispersions. Consequently, zeta potential serves as an important indicator of the stability of nanoparticle-based therapeutic agents. Zeta potential is measured in millivolts (mV) using the ZetaView x30 instrument.

EV Integrity

This EV Integrity Assay is a fluorescence-based method designed to assess the structural integrity of extracellular vesicles (EVs). By selectively detecting signals from intact, biologically-derived particles, this high-throughput assay provides a reliable and quantitative measure of EV integrity. It is particularly useful for evaluating EV stability under different purification conditions, in different formulation buffers, and under varying storage temperatures. Results are reported as fluorescence activity per 1 × 10⁹ particles.

Macrophage Immunomodulation

This macrophage immunomodulation assay offers a powerful tool to assess the ability of the test sample to modulate the inflammatory response of pro-inflammatory macrophages. Nitric oxide production, a key marker of macrophage activation, is quantified to assess phenotypic shifts following treatment. Results are reported as the percent change in nitric oxide levels relative to negative control, with a reduction indicating a potent anti-inflammatory effect of the test sample. Additional readouts are available upon request to further characterize macrophage responses.

RNA Sequencing (RNA-seq)

High-resolution RNA profiling is a core analytical method for characterizing extracellular vesicle (EV) preparations and linking RNA content to biological activity. Using next-generation sequencing (RNA-seq), the assay provides sensitive, reproducible measurement of microRNAs (miRNAs) and other small RNAs, enabling both relative comparisons and quantitative assessment across sample types and manufacturing processes.