Fluorescent Dyes-Based Exosomes Labeling Service

Exosomes, as nano-scale particles, need to be labeled to observe the traces of exosomes in vivo and in vitro. For the study of exosome function, it is generally necessary to observe whether the corresponding phenotypic indicators in the recipient cells, tissues, or organs change after the exosomes are taken up by the recipient cells or reach the target tissues and organs. Therefore, exosome labeling is the basis and key step for the study of exosome function. Creative Biolabs has a complete technical platform and rich service experience and can provide a variety of commonly used fluorescent dyes for labeling exosomes, including carbocyanine dyes, PKH dyes, and penetrant dyes.

In the exosome-related articles published so far, most of the exosomes are labeled with lipophilic dyes. The specific labeling principle is that the lipophilic fluorescent dye embeds its aliphatic tails into the phospholipid biomolecules of exosomes in a non-covalent manner, thereby emitting a stable and long-lasting strong fluorescent signal.

Lipophilic Carbocyanine Dyes for Labeling Exosomes

DiL, DiR, DiO, and DiD are a series of lipophilic fluorescent carbocyanine dyes with high extinction coefficient, polarity dependence, and short excited state lifetime. They can be used to stain cell membranes and other lipid-soluble biological structures. The fluorescence emitted by these fluorescent dyes is very weak before entering the exosome membrane. After binding to the exosome membrane and entering the phospholipid bilayer, these fluorescent dyes can diffuse to the surroundings. When these fluorescent dyes reach the optimal concentration, the entire membrane structure of exosomes can be labeled, and the fluorescence intensity of these fluorescent dyes is greatly enhanced. In addition, the background of the fluorescence imaging of lipophilic carbocyanine dyes is clean, which can avoid background interference caused by autofluorescence in the visible light range in organisms.

Fig.1 The uptake of Dio-Nor-Exos and Dio-Hypo-Exos by HUVECs. (Yang, 2022)

Lipophilic PKH dyes for Labeling Exosomes

PKH dyes were invented by Paul Karl Horan. They have the characteristics of less toxicity, low fluorescence background, high fat solubility, strong penetrability, and strong and stable fluorescence. Unlike carbocyanine dyes, PKH dye molecules have longer aliphatic tails that can be inserted into phospholipid bilayers, while fluorophores stay outside the exosome membrane and emit strong fluorescence. PKH dyes commonly used for exosome labeling include PKH-67 and PKH-26. The in vivo fluorescent half-life of PKH67 is 10-12 days. Compared with PKH-67, PKH-26 has a longer half-life (up to 100 days or more) due to its stronger fluorescence stability.

Fig.2 PKH26-labeled Exos (red) and DAPI (blue). Arrows indicate Exos. (Chen, 2020)

In addition to common lipophilic dyes, penetrant dyes can pass through the membrane structure of exosomes and enter the interior of exosomes.

Penetrating dyes for Labeling Exosomes

Commonly used penetrating dyes are carboxyfluorescein diacetate succinimidyl ester (CFSE) and calcein acetyl methyl ester (Calcein-AM). CFSE itself has no color and fluorescence, and can passively diffuse into exosomes. Until the acetate group of CFSE is removed by the esterase in exosomes, CFSE can react with intracellular amino groups to form a stable fluorescent conjugate. Calcein-AM introduces acetoxymethyl (AM) groups based on traditional Calcein to increase hydrophobicity so that it can easily penetrate the exosome membrane. Once in exosomes, Calcein-AM is cleaved by intracellular esterases to form impermeable Calcein, which is trapped in cells and emits strong green fluorescence. The integrity of exosomes can be detected by Calcein-AM staining. If the integrity of the exosomes is good, Calcein that enters the interior of the exosomes can remain in the exosomes and emit green fluorescence. Otherwise, it will leak out from exosomes.

Fig.3 CFSE-labeled exosomes localize in maternal and fetal tissues. (Sheller-Miller, 2019)

Excitation and Emission Wavelengths of Commonly Used Fluorescent Dyes

Fluorescent dyes	Observed Color	Marked location	Excitation Wavelength	Emission Wavelength
DiO	Green	Intramembrane	484 nm	501 nm
DiL	Orange	Intramembrane	549 nm	565 nm
DiD	Red	Intramembrane	644 nm	665 nm
DiR	Deep Red	Intramembrane	750 nm	780 nm
PKH-67	Green	Extramembrane	484 nm	501 nm
PKH-26	Red	Extramembrane	551 nm	567 nm
CFSE	Green	Inside	494 nm	521 nm
Calcein-AM	Green	Inside	490 nm	515 nm

The above-mentioned commonly used fluorescent dyes for labeling exosomes can be used for in vivo and in vitro functional studies of exosomes, such as exosome tracking services such as exosome labeling, in vitro cell uptake, and in vivo animal imaging. Creative Biolabs has launched various exosome dye products and exosome tracing services. You can contact us to put forward your needs according to your research purposes. We will make the most suitable plan for you.

References

Yang, ZJ.; Bi, QC.; et al. Exosomes Derived from Glioma Cells under Hypoxia Promote Angiogenesis through Up-regulated Exosomal Connexin 43. International Journal of Medical Sciences. 2022. 19(7):1205-1215.
Chen, HX.; Liang, FC.; et al. Exosomes derived from mesenchymal stem cells repair a Parkinson's disease model by inducing autophagy. Cell Death & Disease. 2020. 11(4):288.
Sheller-Miller, S.; Trivedi, J.; et al. Exosomes Cause Preterm Birth in Mice: Evidence for Paracrine Signaling in Pregnancy. Scientific Reports. 2019. 9(1):608.