AAT Bioquest


Illustration of endosome stages
Illustration of endosome stages, including pathways to lysosome fusion and exosome development. Figure made in BioRender.
Exosomes are small, extracellular vesicles (EVs) that have proved to be critical components in cell messaging and as carriers of various biomolecules from one cell to another, influencing a broad range of physiological processes. Initially thought to be a waste byproduct of a cell upon initial discovery in the 1980's, exosomes are now recognized as a leading mechanism in cell-to-cell communication. Ranging from the treatment of diseases to the next big breakthrough in skin and hair, exosomes are largely studied for their vast capabilities of removal of unnecessary cellular components and the delivery of others. Exosomes are a mediating mechanism in communication amongst cells, with extensive research dedicated to the capabilities of what they transport into and out of a cell.

As nanoparticles, exosomes range in size from 30-150 nm, approximately 1/1000 of the size of a cell. They are transporters of cell-specific lipids, proteins, amino acids, metabolites, and genetic material that are necessary to surrounding cells, creating a major role in the recipient cell's programming. Because of the different bioparticles being transported, exosomes have potential to provide information on a plethora of cell functions, processes and in use as biomarkers for targeted therapies.


Exosomes & Cellular Activity

To understand exosomes, we must first understand endosomes. Endosomes are intracellular vesicles (ICV) from which exosomes are created. An inward invagination of a cell's membrane creates the ICV, where it will collect the necessary biomolecules through the endosome membrane thus creating an exosome inside, which will later be released into the extracellular fluids to fuse with a target cell.

The process by which secretory vesicles fuse with the plasma membrane is called exocytosis, expelling necessary components into the extracellular fluid (ECF). Alternatively, endocytosis is the releasing of contents, or the engulfing of the vesicle, into another cell. Genetic material such as DNA, messenger RNA (mRNA) and microRNA (miRNA), along with surface proteins and lipids come together in this process, fusing inside of the lipid vesicle, with specific predetermined targets outside of the cell to carry out regulation, growth, immunity and more. As the cell components fuse into the endosome, some of the membrane is taken up which creates the membrane of the exosome. Furthermore, compartments inside the endosomes sort the various proteins into additional vesicles, called intraluminal vesicles (ILVs), creating what is called multivesicular bodies, or MVBs. It is the fusion of these MVBs with the plasma membrane of a cell that will eventually secrete the exosome.

Endosomes exist in a few different stages but the two most prominent in exosome creation are early endosomes, which become early-sorting endosomes (ESEs) that mature into late endosomes, the secondary, containing the MVBs. Within the original cell, endosomes can go through several pathways, recycling and sorting vesicles and components to the nucleus, via the trans-Golgi network, into lysosomes for removal, or as future exosomes.


Illustration of the basic aspects and general composition of an exosome, showing extracellular matrix proteins and receptors, as well as common enclosed materials; the 'cargo'. Figure made in BioRender.
Blebbing off from the original cell, an exosome protects the cargo inside from degradation that could occur within the ECF. It will then do one of three things, either fuse with the target cell membrane, penetrate and be engulfed by its target cell (where it will release the proteins, lipids, DNA/RNA that are inside, achieving intercellular signaling), or attach directly to the target cell's membrane. The uptake of exosomes into a cell involves specialized receptor interactions, ultimately resulting in the change or fate of that cell. Adhering to the cell membrane itself, “docking”, results in downstream signaling and includes various receptors such as tetraspanins, glycoproteins, adhesion molecules, antigen-presenting molecules and other signaling receptors.

If fusing with the cell membrane, the defined cargo will be released into the cytosol of the cell. Internalization of the exosome into the cell results in two pathways; one pathway is delivery, where the cargo will go directly to the necessary target. The other is endosomal, where the exosome enters the early endosome stage, and later breaks down for the overall health of the recipient cell. Each transaction is highly specialized for what that cell needs - whether it be to maintain a healthy state of homeostasis, or otherwise. There are a number of factors that determine the abundance of exosomes developed and the nature of their existence. The cell source, including age and gender/sex, health or disease status, and any physiological conditions will affect how and where exosomes occur.

Exosomal delivery is highly studied today. The application of EVs features their use as biomarkers, in therapy and drug delivery, for vaccines, and in cosmetics. Scientists are able to produce and harvest exosomes in the lab, loading them with medicine and directly injecting them into a patient to target precise drug delivery. Exosomes can display an affinity for a certain type of tissue, making this targeted technique extremely useful.


Exosome Processing

Exosomes from 100,000 HeLa cells
Exosomes from 100,000 HeLa cells (cultured for 16 hours in 10 mL serum-free medium) were isolated using ReadiPrep™ Exosome Isolation Kit and detected using the Cell Navigator™ Flow Cytometric Exosome Staining Kit.
Though more research must continue to be done to streamline standardization across research groups, clinical advancements are abundant thus far and the potential that exosomes present allows for much more to be explored. Isolation, detection, and analyzation of exosomes are ongoing fields of study, and can be done via a few different methods, with the source of exosomes coming from bodily fluids or supernatant cells.

To isolate, scientists use techniques such as immunoaffinity capture (beads), precipitation, differential centrifugation and/or size exclusion filtration, each of which comes with its advantages and disadvantages. One of the oldest, most-used methods is the use of immunoaffinity beads, typically coated with specific antibodies that bind to exosomes. The most common marker proteins used are tetraspanins CD9, CD63 and CD81, with some use of CD37, CD53 and CD82. These tetraspanins are easy to identify and are capable of separating EVs from complex samples and yield the highest purities.

Once the exosomes are bound to the conjugates, the RNA, DNA, or various proteins can be isolated, or more antibodies can be added to the exterior of those exosomes attached to the magnetic bead and then flow cytometry can be carried out. Precipitation provides a quick method of isolating exosomes but does not provide great purity. At this level, both DNA and RNA can be isolated. Differential centrifugation is found to be one of the best methods to isolate exosomes, especially in the way of purities, although it is time-consuming. Finally, size-exclusion filtration proves to be the best and purest technique by use of chromatography, but it is not quantitative and produces a lower yield. Knowing the distinct pore size of a membrane and filtering out exosomes in this way is an alternative approach, but clogging tends to be a common issue, and it is time-consuming.

FAQs: Digital Catalogs:


Exosome Visualization

iFluor® 555-Wheat Germ Agglutinin (WGA) Conjugate
Live HeLa cells were stained with iFluor® 555-Wheat Germ Agglutinin (WGA) Conjugate (red) at 5 µg/mL for 30 minutes followed by Hoechst 33342 (blue). Image was acquired using fluorescence microscopy using Cy3/TRITC and DAPI filter set.
Staining agents commonly used are fluorescent conjugates such as Wheat Germ Agglutinin (WGA), Annexin V, Cholera Toxin Subunit B (CTB), and Concanavalin A (ConA), each of which is a carbohydrate-binding protein that carries its own affinity for a reaction, be it red-blood cell labeling, apoptosis detection, or glycoprotein binding. Membrane labeling can be done by use of lipophilic fluorescent dyes (carbonaceous, FM, PKH), and the formation of a membrane analog, effectively marking the membrane components. Which staining agents to use largely depends on the cell type being analyzed and the occurrence or distribution of each target.

Quantification of exosomes will continue to be an active discipline of study amongst researchers, to detect the slightest of pathological changes with minimal preparation of a given sample, and with high yield and purity. Currently, technologies being used to quantify exosomes look at factors such as the stoichiometry of miRNAs within each exosome. Advanced nanoparticle tracking analysis uses light scattering, which determines the size and count of exosomes within a suspension flow chamber. Another method called Tunable Resistive Pulse Sensing (TRPS) provides information on the concentration and size of the exosomes in solution through a distinct nanopore. Other techniques include surface plasmon resonance, where concentration of EVs in solution can be determined by use of surface-based sensors, and enzyme-linked immunosorbent assay, or ELISA, a broader look at the specific markers associated with particular materials inside the exosome.

Further analyzation of EVs can be accomplished by methods such as the Western blot, mass spectrometry, electron microscopy, or flow cytometry. Like isolation, the ways in which scientists can analyze exosomes have their benefits and drawbacks. With Western blots, though providing the presence of EV markers, researchers are unable to look at proteins at a single level and this technique requires large samples and can be laborious. Mass spectrometry is also unable to be analyzed at a single level, is more sensitive and requires expertise in the set-up and operation. Electron microscopy is useful in immunostaining but has its challenges and can be studied only a few at a time.

Flow cytometry, by far the most robust of the commonly used platforms, allows for the study of multiple surface markers on EVs to be screened at one time, is quick and widely available, and goes as far as measuring the individual properties of the particles involved. Like everything else in a lab setting, there is room for error and results can be affected by less suitable storage methods or contamination issues.

Table 1. iFluor® wheat germ agglutinin conjugates for live and fixed cells.

Product name
Ex/Em (nm)
Filter Set
Unit Size
Cat No.
iFluor® 350-Wheat Germ Agglutinin ConjugateMembrane-impermeant345/450DAPI1 mg25525
iFluor® 488-Wheat Germ Agglutinin ConjugateMembrane-impermeant491/516FITC1 mg25530
iFluor® 532-Wheat Germ Agglutinin ConjugateMembrane-impermeant537/560Cy3/TRITC1 mg25532
iFluor® 555-Wheat Germ Agglutinin ConjugateMembrane-impermeant557/570Cy3/TRITC1 mg25539
iFluor® 594-Wheat Germ Agglutinin ConjugateMembrane-impermeant588/604Texas Red1 mg25550
iFluor® 647-Wheat Germ Agglutinin ConjugateMembrane-impermeant656/670Cy51 mg25559
iFluor® 680-Wheat Germ Agglutinin ConjugateMembrane-impermeant684/701Cy51 mg25560
iFluor® 700-Wheat Germ Agglutinin ConjugateMembrane-impermeant690/713Cy51 mg25561
iFluor® 750-Wheat Germ Agglutinin ConjugateMembrane-impermeant757/779Cy71 mg25562
iFluor® 790-Wheat Germ Agglutinin ConjugateMembrane-impermeant787/812Cy71 mg25563



MSCs-ApoEVs promote fusion and apoptosis ratio of C2C12 myoblasts in vitro. The representative fluorescence images of C2C12 myoblasts after MSCs-ApoEVs treatment, MSCs-ApoEVs were pre-stained by PKH26, actin by Phalloidin-iFluor® 488 conjugate, and nuclei by Hoechst. Scale bar indicates 50 μm. Source: MSCs-derived apoptotic extracellular vesicles promote muscle regeneration by inducing Pannexin 1 channel-dependent creatine release by myoblasts by Qingyuan Ye, Xinyu Qiu, Jinjin Wang, Boya Xu, Yuting Su, Chenxi Zheng, Linyuan Gui, Lu Yu, Huijuan Kuang, Huan Liu, Xiaoning He, Zhiwei Ma, Qintao Wang & Yan Jin. International Journal of Oral Science, January 2023.
The ways in which exosomes have proven to be beneficial and a breakthrough in scientific research are plentiful. As messenger vesicles, their components can activate one's immune system, produce B-cells and helper T-cells, generating anti-inflammatory responses thanks to the correspondence of those proteins that down-regulate inflammatory proteins, as well as improve brain plasticity and neuron stress tolerances.

Exosomes are capable of regenerating tissue when secreted from stem cells, with over 1000 growth factors observed, amplifying the possibilities of studies related to stem cell research. In the way of health and beauty, exosomes are used to help rebuild collagen and elastin, strengthening skin and providing the much-desired “glow” amongst consumers. It is the regenerative qualities and capabilities of exosomes that have brought exosomes to the forefront of many new advancements and possibilities of studies.

Some of the negative roles that exosomes play include the spread of viruses (i.e.: Hepatitis B), their association with apoptosis in T-cells resulting in immune cell deficiencies, and the eventual development of different neurological disorders is linked to the spread of exosomes within the body, such as Parkinsons and Alzheimer's. Moreover, their involvement in heart failure, liver disease, diabetes, and cancer has also been observed. Tumor cells have been studied and proven to release exosomes that contain cancer signals that spread throughout the body. On the other hand, tumors secreting exosomes can also be beneficial in signaling to immune cells and inducing death in harmful cells.

With much more diagnostical research to still be done, additional advancements in the knowledge and understanding of how exosomes function, their heterogeneity, and the importance of their specialized cargo, will continue to be made.

Table 2. DiI, DiO, DiD and DiR dyes

Product Name
Ex (nm)
Em (nm)
Unit Size
22042DiOC16(3) perchlorate [3,3-Dihexadecyloxacarbocyanine perchlorate]48350125 mg
22102DiI perchlorate [1,1-Dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate] *CAS 41085-99-8*500564100 mg
22034DiD Perchlorate *UltraPure Grade*64666325 mg
22070DiR iodide [1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide]75477825 mg

Product Ordering Information

Table 3. Exosome Kits

Product Name
Unit Size
22426Cell Navigator™ Flow Cytometric Exosome Staining Kit100 Tests
60204ReadiPrep™ Exosome Isolation Kit50 Preps
60205ReadiPrep™ Exosome Isolation Kit200 Preps

Table 4. Lipophilic tracers for labeling cell membranes in live and fixed cells and tissues.

Product Name
Ex/Em (nm)
Filter Set
Unit Size
Cat No.
DiOC2(3) iodide [3,3-Diethyloxacarbocyanine iodide]482/500FITC25 mg22038
DiOC3(3) iodide [3,3-Dipropyloxacarbocyanine iodide]482/500FITC25 mg22039
DiOC7(3) iodide [3,3-Diheptyloxacarbocyanine iodide]482/500FITC25 mg22040
DiOC16(3) perchlorate [3,3-Dihexadecyloxacarbocyanine perchlorate]482/500FITC25 mg22042
DiOC5(3) iodide [3,3-Dipentyloxacarbocyanine iodide]482/500FITC25 mg22045
DiOC6(3) iodide [3,3-Dihexyloxacarbocyanine iodide]482/500FITC25 mg22046
DiO perchlorate [3,3-Dioctadecyloxacarbocyanine perchlorate]482/500FITC25 mg22066
CytoTrace™ CM-DiI548/563TRITC10x50 µg22057
DiIC12(3) perchlorate [1,1-Didodecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate]549/563TRITC25 mg22035
DiIC16(3) perchlorate [1,1-Dihexadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate]549/563TRITC25 mg22044
DiIC12(3)-DS [1,1-Diododecyl-3,3,3,3-tetramethylindocarbocyanine-5,5-disulfonic acid]549/563TRITC5 mg22050
DiIC18(3)-DS [1,1-Dioctadecyl-3,3,3,3-tetramethylindocarbocyanine-5,5-disulfonic acid]549/563TRITC5 mg22052
DiSC2(3) [3,3-Diethylthiacarbocyanine iodide]560/571TRITC25 mg22073
DiI iodide [1,1-Dioctadecyl-3,3,3,3- tetramethylindocarbocyanine iodide]549/563TRITC100 mg22101
DiI perchlorate [1,1-Dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate]549/563TRITC100 mg22102
DiI triflate [1,1-Dioctadecyl-3,3,3,3-tetramethylindocarbocyanine triflate]549/563TRITC100 mg22103
DiA [4-(4-(Dihexadecylamino)styryl)-N-methylpyridinium iodide]491/611Texas Red25 mg22030
CytoTrace™ CM-DiD643/663Cy510x50 µg22059
DiD labeling solution [1,1-Dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine]645/663Cy510 mL22033
DiD Perchlorate *UltraPure Grade*646/663Cy525 mg22034
DiIC12(5)-DS [1,1-Diododecyl-3,3,3,3-tetramethylindodicarbocyanine-5,5-disulfonic acid]650/670Cy55 mg22051
DiIC18(5)-DS [1,1-Dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine-5,5-disulfonic acid]652/668Cy55 mg22054
DiIC1(5) iodide [1,1,3,3,3,3-Hexamethylindodicarbocyanine iodide]640/657Cy525 mg22056
DiSC3(5) [3,3-Dipropylthiadicarbocyanine iodide]660/675Cy525 mg22076
DiR iodide [1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide]754/778Cy725 mg22070



The biology, function, and biomedical applications of exosomes
Exosomes: biogenesis, biologic function and clinical potential
The exosome journey: from biogenesis to uptake and intracellular signalling
What Are Exosomes? | Clinical Applications
The Science of Exosomes
Exosome Quantification