Labeling and Identification of Exosomes

In 2014， the International Society for Extracellular Vesicles (ISEV) provided researchers with a minimal set of biochemical, biophysical, and functional standards that should be used to attribute any specific biological cargo or function to extracellular vesicles (EVs). ISEV suggests a format of characteristics of EVs that should be analyzed and at least 2 different technologies should be used to characterize individual EVs.

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■ Detection of exosome-enriched proteins

Analytic techniques to detect exosome-enriched proteins include #Westernblot (WB), high-resolution flow cytometry (FACS), or global proteomic analysis using mass spectrometry to identify exosome transporting-related transmembrane proteins (CD63/CD81/CD9), heat shock proteins (HSP60/HSP70/HSPA5/CCT2/HSP90) and some cell-specific proteins that are enriched in exosomes membrane. Among them, #CD63 and TSG101 are the most commonly used exosome markers.

■ Identification of exosome morphology with #TEM

Exosome morphology can be directly identified under transmission electron microscopy (TEM) with high resolution to provide an indication of the heterogeneity of the samples.

■?Size distribution and concentration measurements with NTA

#NTA (Nanoparticle Tracking Analysis) technology can quickly and accurately analyze the particle size distribution and concentration of a large number of exosomes in the sample.

A study by Muyu Yu et al. focused on whether exosomes derived from the bone marrow MSC (BMSC) pretreated with Atorvastatin(ATV) could exhibit better pro-angiogenic ability. As shown in Fig 4, they isolated exosomes from non-pretreated BMSC (Exos) and ATV-pretreated BMSC (ATV-Exos) and evaluated their characterization by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting[7].?

■ Labeling and tracing of #exosomes

In vitro?labeling or in vivo tracing of isolated exosomes is helpful for further studies on the functions and applications of exosomes. There are many methods for labeling and tracing exosomes, mainly including lipophilic tracers and membrane-permeable compound tracers.

Lipophilic Tracers

Lipophilic dyes (DiO, DiI, DiD, DiR) can label exosomes well and are also commonly used dyes in exosome research.

In vitro?tracing: Exosomes can be traced in vitro by using PKH26 dye and scanning it in a confocal microscope. As shown in Fig. 5, PKH26-labeled exosomes (red) were localized in the perinuclear region of HUVEC by using laser scanning confocal microscopy, confirming the internalization of exosomes by endothelial cells[7].?

In vivo?tracing:

In vivo tracing can be done by using DIR?dye and scanning by IVIS 200 Optical Imaging System.

Membrane permeable compound tracers

The membrane-permeable?carboxyfluorescein diacetate succimidyl ester (CFSE) is a lipophilic fluorescent dye that can passively diffuse into cells and is also commonly used for the effective tracing of exosomes. It is colorless and non-fluorescent until the acetate groups are cleaved by intracellular esterases to yield highly fluorescent carboxyfluorescein succinimidyl ester.?The succinimidyl ester group reacts with intracellular amines forming fluorescent conjugates that are well retained by cells.

MCE has launched multiple exosome-related products. Please check our website for specific product information.

References

[1] Cell Biosci. 2019 Feb 15;9:19. ?

[2] Diabetologia. 2019 Dec;62(12): 2179-2187.

[3] APL Bioeng. 2019 Mar 27;3(1):011503?

[5] Nat Biomed Eng. 2022 Jul 4

[6] Front Bioeng Biotechnol. 2022 Jan 5;9:811971?

[7] Stem Cell Res Ther. 2020 Aug 12;11(1):350.

[8] Control Release. 2015 Feb 10; 199: 145-55.

[9] Sci Rep. 2019 Jan 24;9(1):608.

Related Products

HY-K1062 Exosome extraction and purification kit (from cell culture media)

HY-K1063 Exosome extraction and purification kit (from serum or plasma)

HY-K1065 Exosome extraction and purification kit (from body fluids)

HY-K1064 Exosome CD63 and TSG101 Detection Kit

HY-133876 DiA

HY-D0969 DiO

HY-D1028 DiD perchlorate

HY-D0938 CFSE

All MCE products are only used for scientific research or drug?registration?applications, we do not provide products and services for any personal use.