Marking vesicles without over-claiming what they are: why a cd81 antibody and a cd63 antibody detect tetraspanins that are enriched on small vesicles rather than exclusive to them, what an exosome antibody or the exosome antibodies sold as a set can honestly establish about origin, how an annexin a1 antibody and an annexin a2 antibody distinguish populations that a tetraspanin panel merges, why a preparation has to be characterised by more than one marker and by a negative marker as well, and what a calreticulin antibody run alongside is doing as a contamination control rather than as a vesicle marker

Characterising a preparation

1. Use at least three positive markers. Single marker characterisation is not accepted, because each marker is present on a subset and on other material. A panel spanning different marker classes describes the preparation far better than any one of them.
2. Include a negative marker. An abundant intracellular protein from a compartment that should not be in the preparation shows whether cell debris or lysed material contaminates it. This is the control that most preparations lack and most reviewers ask for.
3. Report the isolation method with every result. Differential centrifugation, density gradient, size exclusion and precipitation yield different populations with different contaminants. Two preparations by different methods are not comparable, and the method belongs in the figure legend.
4. Measure particle number and protein separately. Protein per particle differs enormously between preparations and between methods. Reporting only protein makes a contaminated preparation look abundant.
5. Do not infer origin from markers alone. Marker enrichment supports a claim about vesicle type; it does not establish biogenesis route. Where origin matters, genetic perturbation of the pathway is what supports it.

Enrichment is the honest word

The vocabulary in this field runs ahead of the evidence, and marker panels are frequently presented as identifying a biogenesis route they cannot establish. Writing enrichment where enrichment is what was shown costs nothing and makes the work durable.

Community guidance in this area has converged on multiple positive markers, at least one negative marker and full reporting of the isolation method. Following it removes the most common criticism of these papers.

The preparation is the experiment

More variance in vesicle work comes from isolation than from detection. Serum in the culture medium contributes its own particles, cell viability changes what is released, and centrifugation conditions select different populations.

Use depleted medium, record viability, fix one method and report it. Those four steps account for most of the reproducibility difference between laboratories.

Common questions

Is a tetraspanin stain proof of exosomes?

No. Those markers are enriched on small vesicles and are also found on the plasma membrane and other particles. They support a claim of enrichment, and origin needs perturbation of the biogenesis pathway.

Which negative marker should be used?

An abundant protein from a compartment that should be absent, run on the same blot with equal loading. Its absence is what distinguishes a vesicle preparation from a preparation of cell fragments.

Why do preparations differ so much between laboratories?

Because isolation method, starting material and culture conditions all change what is recovered. Reporting the method in full is the minimum requirement for anyone else to interpret the result.

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