Technical reference
MSC Exosome Characterization Guide.
Exosome characterization is the set of analytical methods used to verify that an exosome preparation contains the correct vesicles at the right concentration and purity. This guide covers NTA, TEM, Western blot, flow cytometry, and the standards researchers should expect from a quality supplier.
Why characterization matters
Without characterization data, there is no way to distinguish exosomes from other extracellular vesicles, protein aggregates, or liposomal contaminants. A supplier claiming to sell "exosomes" without NTA, marker confirmation, and endotoxin data is asking researchers to take their word for it — and that word has no analytical backing.
The International Society for Extracellular Vesicles (ISEV) publishes Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines that define the minimum characterization data required. B&H Bio's quality system is designed to meet these expectations.
Core characterization methods
1. Nanoparticle Tracking Analysis (NTA)
NTA measures particle size distribution and concentration by tracking the Brownian motion of individual particles in suspension using laser light scattering. It is the standard method for quantifying exosome size and concentration.
| NTA Parameter | Expected Range | What it tells you |
|---|---|---|
| Mean particle size | 50-150 nm | Confirms vesicles are in exosome size range |
| Mode particle size | 50-120 nm | Most common particle size in distribution |
| Particle concentration | Reported in particles/mL | Quantifies exosome yield per volume |
| Size distribution profile | Single peak, narrow distribution | Broad or multi-peak suggests contamination |
2. Transmission Electron Microscopy (TEM)
TEM provides direct visual confirmation of exosome morphology. Exosomes typically appear as cup-shaped or spherical vesicles with a lipid bilayer membrane. TEM complements NTA by verifying that the particles measured by NTA are actually vesicles.
Key TEM verification points: presence of lipid bilayer, cup-shaped/spherical morphology, size consistent with NTA data, absence of large debris or non-vesicular particles.
3. Western Blot for Exosome Markers
Western blot confirms the presence of exosome-enriched protein markers. The ISEV MISEV guidelines recommend testing for:
| Marker | Category | What it confirms |
|---|---|---|
| CD9 | Tetraspanin (transmembrane) | Exosome-enriched surface protein |
| CD63 | Tetraspanin (transmembrane) | Exosome-enriched surface protein |
| CD81 | Tetraspanin (transmembrane) | Exosome-enriched surface protein |
| TSG101 | ESCRT machinery (cytosolic) | Exosome biogenesis pathway protein |
| Alix | ESCRT-associated (cytosolic) | Exosome biogenesis pathway protein |
Negative controls should also be reported — markers that should NOT be present in exosome preparations, such as calnexin (ER marker), GM130 (Golgi marker), or cytochrome c (mitochondrial marker). Presence of these indicates cellular contamination.
4. Flow Cytometry
Flow cytometry can be used to quantify exosome surface markers at the single-vesicle level. While traditional flow cytometers struggle with the small size of exosomes, specialized high-resolution flow cytometry and bead-based flow cytometry methods can provide quantitative marker data.
5. Protein Quantification
Total protein content (BCA or Bradford assay) should be reported alongside particle concentration. The particle-to-protein ratio helps assess purity — a high protein content relative to particle count suggests protein contamination.
Quality markers researchers should expect
| Quality Indicator | What to expect from a quality supplier | Red flag |
|---|---|---|
| NTA data | Size distribution graph + concentration | "Characterized" with no data |
| Exosome markers | CD9+, CD63+, CD81+ confirmed | No marker data |
| Negative controls | Calnexin/GM130 negative | No negative controls tested |
| Endotoxin level | Specific value (e.g., <0.5 EU/mL) | "Endotoxin-free" without data |
| Sterility | USP <71> or equivalent method | "Sterile" without method |
| Protein content | Reported alongside particle count | Only particle count, no protein data |
B&H Bio's characterization standards
B&H Bio's exosome quality records include:
- Particle characterizationNTA data showing size distribution and concentration for every lot.
- Exosome markersCD9, CD63, CD81 confirmation as part of the quality documentation.
- Endotoxin testingUSP <85> endotoxin criteria with reported values.
- Sterility testingUSP <71> sterility criterion.
- Third-party verificationEurofins-verified COA for every batch — independent lab confirmation.
- Lot traceabilityEvery lot reconcilable with its characterization data.
Review the data
Request characterization records.
NTA, markers, endotoxin, sterility — review the actual data for our exosome materials.