Flow Cytometry for Exosome Detection: Methods, Strategies, and Experimental Tips

1. What Are Exosomes?

Exosomes are nanoscale extracellular vesicles (EVs) secreted by cells, typically ranging from 30–150 nm in diameter. Their size is comparable to certain viruses, although they lack autonomous replication capacity. In essence, exosomes are secretory vesicles that act as cellular “nano-messengers”. They possess a bilayer membrane and encapsulate a rich cargo of biomolecules—including nucleic acids, proteins, and lipids—that enable precise intercellular molecular transfer and communication.

In 2013, the Nobel Prize in Physiology or Medicine was awarded to James Rothman, Randy Schekman, and Thomas Südhof for their seminal work on vesicle trafficking mechanisms, which brought exosome research into the spotlight.

Exosomes originate from most cell types as intraluminal vesicles formed by inward budding within multivesicular bodies (MVBs). When MVBs fuse with the plasma membrane, these vesicles are released as exosomes. Once secreted, they can transfer DNA, RNA, and proteins to recipient cells, influencing cellular behaviour and diverse physiological processes.

Exosomes are widely present in cell culture supernatants and various biological fluids such as blood, lymph, saliva, urine, semen, and breast milk. They are also found in tissue-derived samples, including brain, muscle, and adipose tissue.

Figure 1. Structure of exosomes (DOI:10.3390/biology10040285)

2. Flow Cytometry Approaches for Exosome Detection

The nanoscale dimensions of exosomes exceed the optical detection limits of conventional flow cytometers. Therefore, specific strategies are required for reliable analysis. Current mainstream approaches include:

2). Bead-based Amplification Detection

Exosome-specific antibodies (e.g. CD9, CD63, CD81) are pre-coated onto microspheres. These beads capture exosomes, which are then detected using fluorochrome-labelled secondary antibodies (targeting another exosomal surface antigen or the host species of the primary antibody). Flow cytometry is subsequently employed to analyse the bead–exosome complexes. This approach enhances detection accuracy and is compatible with most standard flow cytometers.

Figure 2. Principle of bead-based exosome detection(DOI:10.1016/j.jddst.2021.102526)

2). Nano-flow Cytometry

High-sensitivity instruments (e.g. Beckman CytoFLEX nano, ApogeeFlow MicroPLUS / Micro) allow direct detection of exosomes. These cytometers feature optimised optics, detectors, and software, enabling the acquisition of measurable scatter or fluorescence signals from individual nanoparticles (tens to hundreds of nanometres). This makes it possible to perform single-particle counting, size estimation, and surface marker analysis.

Figure 3. Flow cytometric characterisation of exosome size and surface markers (image source: Beckman website)

A. Standardised microspheres for calibration. B. Exosome size distribution. C. Size distribution after Triton X-100 treatment. D. Expression analysis of PE-CD81 or APC-CD63-labelled exosomes.

3. Practical considerations for flow cytometric detection of exosomes

1). Thorough removal of impurities: Prior to acquisition, remove cell debris and large particles by low-speed centrifugation or filtration to reduce background noise.

2). Use of bright fluorochromes: Employ antibodies conjugated with high-intensity fluorochromes such as PE or APC, and use bright membrane-binding dyes (e.g. PKH, DiI) to “light up” exosomes. Ensure removal of excess dye to prevent false positives.

3). Sample dilution: Exosomes are prone to the “swarm effect”, where multiple small particles are recorded as a single event. Proper dilution and assessment of signal linearity help avoid this artefact.

4). Appropriate controls:

① Unstained control: for voltage adjustment and defining negative populations.

② Isotype control: to discriminate specific from non-specific binding.

③ Fluorescence Minus One (FMO): essential for multicolour experiments.

④ Triton X-100 lysis control: disruption of the exosomal membrane should significantly reduce or abolish true positive signals, providing critical evidence of signal specificity.

5). Instrument calibration and standardisation: Fluorescent calibration beads of known size and concentration should be used to standardise cytometer performance, allowing more reliable estimation of exosome size and abundance.

4. Alternative Methods for Exosome Detection

In addition to flow cytometry, exosomes can be analysed by several other techniques:

1). Fluorochrome labelling and tracking: Lipophilic dyes (e.g. PKH67, PKH26, DiI, DiD, DiO) stably incorporate into the lipid bilayer, enabling visualisation of exosome uptake, biodistribution, and homing via confocal microscopy, high-content imaging, or in vivo imaging.

2). Electron microscopy (TEM/SEM): Direct observation of vesicle morphology, size, and structural features.

3). Western blotting: Detection of exosomal marker proteins (e.g. CD9, CD63, CD81, TSG101, Alix).

4). ELISA: Quantitative detection using exosome-specific capture antibodies, suitable for targeted analysis.

5. Recommended Exosome Antibodies for Flow Cytometry (abinScience)

abinScience flow cytometry antibodies cover commonly used markers with a broad product range, supporting multi-species research with stable and reliable performance. For the full antibody list, please visit: http://www.abinscience.com

References

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[7] Kadbhane, A., Patel, M., Srivastava, S., Singh, P.K., Madan, J., Singh, S.B., & Khatri, D.K. (2021). Perspective insights and application of exosomes as a novel tool against neurodegenerative disorders: An expository appraisal. Journal of Drug Delivery Science and Technology, 102526.