How to Validate Exosome Integrity After Purification?
- A single and narrow peak indicates relatively high vesicle purity
- The presence of multiple size peaks or particles significantly larger than 150 nm suggests potential aggregates, microvesicles, or lipid droplet contamination
- An unusually low particle concentration may reflect inefficient isolation or vesicle disruption
- Blurred, collapsed, or deformed vesicle membranes
- The presence of abundant impurities or non-vesicular structures
- Markedly abnormal vesicle diameters or pronounced morphological heterogeneity
- Membrane proteins: CD9, CD63, CD81 (tetraspanin family)
- Endosomal proteins: TSG101, ALIX
- Negative control: Calnexin (an endoplasmic reticulum protein that should be absent in exosome preparations)
- PKH26/PKH67 dyes: labeling of lipid bilayers to monitor membrane distribution and structural stability
- Annexin V binding assays: detection of exposed phosphatidylserine to identify membrane disruption or early apoptotic vesicles
- Flow cytometry combined with antibody labeling: discrimination between intact exosomes and vesicular fragments with semi-quantitative capability
- Compatibility with low-input exosome samples down to 10 μg
- Complimentary quality control evaluation of exosome integrity (including NTA, WB, and TEM)
- Customized exosome omics services for applications such as tumor immunology, autoimmune diseases, and stem cell-derived exosomes
Exosomes are nanoscale vesicles generated through the endosomal pathway and are widely present in various biological fluids, including plasma, urine, saliva, and milk. They have attracted extensive interest due to their important roles in intercellular communication, disease biomarker discovery, and drug delivery. However, functional investigations of exosomes rely fundamentally on the preservation of their structural and biological integrity. Loss of integrity may introduce experimental bias and even lead to erroneous conclusions. Therefore, establishing a scientific and systematic strategy for exosome integrity validation is a critical prerequisite for high-quality exosome research.
Why Is Exosome Integrity So Important?
Exosomes possess a typical lipid bilayer membrane structure with a diameter generally ranging from 30 to 150 nm, and they encapsulate specific proteins, RNAs, and lipids. During extraction and purification, improper handling may easily cause membrane rupture, aggregation, or co-isolation with cellular debris and lipid droplets, thereby compromising the reliability of subsequent functional assays and omics analyses. Consequently, before confirming the successful purification of exosomes, their integrity must be comprehensively evaluated from multiple aspects, including particle size distribution, membrane architecture, marker protein expression, and vesicle morphology.
Four Conventional Methods for Validating Exosome Integrity
1. Nanoparticle Tracking Analysis (NTA): Evaluation of Particle Size and Concentration
The primary criterion for assessing exosome integrity is whether their particle size falls within the expected range. NTA precisely determines particle size distribution and concentration by tracking Brownian motion based on laser light scattering. Authentic exosomes are typically distributed within the range of 30–150 nm.
Key considerations for interpreting NTA results:
By comparison, although dynamic light scattering (DLS) can also provide size information, its limited resolution makes it unsuitable for accurately distinguishing heterogeneous particle populations; therefore, it is not recommended as a stand-alone method for exosome integrity assessment.
2. Transmission Electron Microscopy (TEM): Direct Visualization of Vesicle Structure
TEM is widely regarded as the most reliable approach for confirming exosome morphology and membrane integrity. Under negative staining conditions, intact exosomes typically exhibit a characteristic cup-shaped or bowl-shaped appearance with clearly defined bilayer membranes.
The following features may indicate compromised exosome integrity:
Although TEM does not provide quantitative measurements, it remains indispensable for structural validation and is considered one of the most critical pieces of morphological evidence in exosome-related studies.
3. Western Blot or ELISA: Detection of Marker Protein Expression
Purpose: To verify the expression of exosome-specific proteins and exclude contamination from cellular fragments or other vesicular populations.
Representative exosome markers include:
Western blot and ELISA are both applicable for marker detection. Western blot is widely used and provides semi-quantitative information on protein expression levels, whereas ELISA is more suitable for high-throughput screening.
4. Fluorescent Dyes and Flow Cytometry: Assessment of Membrane Integrity and Permeability
Exosome functionality is highly dependent on the integrity and impermeability of the vesicle membrane. Membrane disruption not only compromises cargo stability but also interferes with cellular uptake and downstream signal transduction.
Commonly used strategies include:
These approaches are particularly suitable for rapid quality control prior to functional experiments, especially in large-scale screening studies.
Relationship Between Exosome Integrity and Mass Spectrometry-Based Proteomics
Exosome integrity not only affects structural features and marker protein expression but also directly determines the reliability of downstream omics analyses. In mass spectrometry-based proteomics, membrane rupture may introduce extracellular contaminant proteins into the sample, thereby distorting protein spectra and misleading differential expression analysis. This issue is particularly critical in data-independent acquisition (DIA) and isobaric labeling-based quantification methods such as TMT, where sample quality directly influences protein identification depth and quantitative reproducibility. Therefore, rigorous validation of exosome integrity prior to mass spectrometry analysis is strongly recommended to ensure structural stability and compositional fidelity.
Professional Exosome Research Solutions Supported by MtoZ Biolabs
In the field of exosome research, MtoZ Biolabs has established an integrated one-stop service platform encompassing exosome isolation and purification, integrity validation, mass spectrometry analysis, and bioinformatics interpretation. By leveraging Orbitrap high-resolution mass spectrometry, standardized proteomics workflows, and a dedicated exosome research team, the company provides clients with highly reliable and reproducible analytical support.
Key service features include:
Exosome integrity represents not only a technical parameter but also a hallmark of scientific rigor. Only when both structural and functional integrity are assured can downstream functional assays, mechanistic investigations, and omics analyses yield reliable and interpretable results. For researchers planning exosome-related studies, MtoZ Biolabs offers tailored end-to-end solutions covering the entire workflow from sample preparation to data analysis.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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