Exosomes are a form of extracellular vesicle (EV), which could contribute to malignant transformation and the metastasis of cancer. Consequently, intercellular communication via exosomes is attracting considerable interest in the scientific community. To shed light on such communication, labeling techniques based on fluorescent dyes have been used. Fluorescent dyes that label the cellular membrane are commonly used for exosome labeling because the lipid bilayer in exosomes is a suitable labeling target. ExoSparkler series can be used for staining of purified exosomal membrane or protein, which allows imaging of labeled exosomes taken up by cells.
ExoSparkler series enables you to observe exosome dynamics more accurately.
Commonly used exosomal membrane dye can cause dye aggregation, exhibiting fluorescent spots that are not derived from exosomes. These dyes can also change the functional properties of exosomes while increasing the background imaging.1,2
The dyes used in ExoSparkler series (Mem Dye-Green, Red, and Deep Red) do not cause aggregation and have little influence on properties of exosomes, allowing a more accurate observation of exosome dynamics.
1) Mehdi Dehghani et al., “Exosome labeling by lipophilic dye PKH26 results in significant increase in vesicle size”.bioRxiv., 2019, doi:10.1101/532028.
2) Pužar Dominkuš P et al., “PKH26 labeling of extracellular vesicles: Characterization and cellular internalization of contaminating PKH26 nanoparticles.” Biochim Biophys Acta Biomembr., 2018, doi: 10.1016/j.bbamem.2018.03.013.
ExoSparkler series does not allow extracellular aggregation
Exosomes stained with ExoSparkler’s Mem Dye-Deep Red or an alternative product (green or red) were added to each well containing HeLa cells. The labeled exosomes taken into HeLa cells were observed by fluorescent microscopy. As a result, extracellular fluorescent spots suspected of dye aggregations were seen in each well containing the exosomes stained with the alternative product (green or red).
Exosomes were purified by ultracentrifugation (10μg exosome protein) and stained with each dye. Labeled exosomes were added to HeLa cells (1.25×104 cells), and the cells were incubated for 24 hours. Cells were washed, and immunofluorescence images showing labeled exosomes were observed.
Mem Dye-Deep Red(Purple): Ex 640nm/Em 640-760nm
Alternative Product “P” (Green): Ex 561nm/Em 560-620nm
Alternative Product “P” (Red): Ex 640nm/Em 650-700nm
Mem Dye-Deep Red and Product “P” (Green and Red) in aqueous solution were analyzed by NTA (nanoparticle tracking analysis) to investigate the generation of aggregates. No aggregation was observed in the experiments with Mem Dyes, although Product “P” (Green and Red) produced dye-to-dye aggregates (100–500 nm size).
Instrument: LM10-HSBFT 14 (Nanosight)
Change the particle size of Mem Dye-solution and Product “P” solution
In Mem Dye-Green, Red, the aggregation of the dye was not confirmed as in Mem Dye-Deep Red.
Mem Dyes have little effect on exosome properties
NTA (nanoparticle tracking analysis) and zeta potential were measured to determine the changes in exosomes of dye-stained with Mem Dye-Deep Red or Product “P” (green or red) or unstained exosomes.
As a result, the Mem-Dye series (green, red, deep red) had little effect on exosome properties.
Effect of the dyes on the particle size of the exosomes
Exosomes were stained with Mem Dye-series (green, red, deep red) and Product “P” (green and red) at a dye concentration of 10 μmol/L in DMSO, the NTA (nanoparticle tracking analysis) of the stained exosomes (as 10 µg protein) was measured.
As a result, Mem Dyes-series did not change number and particle size of the exosomes (bottom left). Conversely, the Product “P” stained exosomes showed the significant changes of particle size and population of the exosomes (bottom right).
Instrument: LM10-HSBFT 14 (Nanosight)
Effect of the dyes on the zeta potentials of the exosomes
Exosomes were stained with Mem Dye-series (green, red, crimson) and Product “P” (green and red) at a dye concentration of 10 μmol/L in DMSO, the zeta potentials of the stained exosomes (as 10 µg protein) was measured.
As a result, product “P”-stained exosomes have lower zeta potential than Mem Dye-stained.
Instrument: Zetasizer Nano ZSP (Malvern Panalytical)
Zeta potentials comparison of dye-stained (Mem-Dye/Product “P”) or unstained exosomes
References) Takashi Shimomura et al., “New Lipophilic Fluorescent Dyes for Exosome Labeling: Monitoring of Cellular Uptake of Exosomes”.bioRxiv., 2020, doi:10.1101/2020.02.02.931295.
Our ExoSparkler Exosome Membrane Labelling Kits provide everything from fluorescence labeling to purification
ExoSparkler series contains filtration tubes available for the removal of dyes unreacted after fluorescence labeling, as well as an optimized protocol for labeling exosomes. Our ExoSparkler series makes it possible to prepare fluorescence labeling of exosomes using the simple procedure.
Comparison of purification methods (removal of unlabeled dyes)
The filtration tubes used to remove unlabeled dyes in this kit can purify exosomes at a higher recovery rate than gel filtration methods.
For the effectiveness of purification using filtration tubes, please see Q&A.
(The filter is colored in the purification after the labeling, Have unlabeled dyes been removed?)
Observe the time-dependent changes in exosomes localization
Exosomes purified by ultracentrifugation (10 µg as protein amount) were stained with Mem Dye-Deep Red (Exosome Membrane Fluorescence Labeling Kit) and added to HeLa cells (1.25×104 cells) stained with lysosome staining dye. The fluorescence images were observed after 1 h and 4 h incubation.
As a result, it was confirmed that the fluorescence puncta (purple) of Mem Dye-Deep Red overlapped with the localization of lysosomes (green) over time (white), and that the localization of exosomes changed in a time-dependent manner.
Mem Dye-Deep Red (Purple): Ex 640 nm/Em 640-760 nm
Lysosome staining dye: Ex 488 nm/Em 490-540 nm
Visualization of EVs uptake via endocytic pathway
Mem Dye-labeled EVs are internalized via endocytosis:
HeLa cells were incubated with 10 μmol/L ECGreen (Code: E296) for 30 min. Then, Mem Dye-Deep Red labeled EVs (quantified as 10 µg of protein) were added to HeLa cells. After 30 or 120 min incubation, the cells were washed and observed under a fluorescence microscope (Scale Bar: 10 µm).
ExoSparkler series product comparison
Exosomes were purified by ultracentrifugation (10 μg exosome protein) and stained with each dye. Labeled exosomes were added to HeLa cells (1.25×104 cells), and the cells were incubated for 24 hours. Cells were washed, and immunofluorescence images showing labeled exosomes were observed.
Green: Ex 488nm/Em 490-540nm
Red: Ex 561nm/Em 570-640nm
Deep Red: Ex 640nm/Em 640-760nm
1) R.Kawata, S.Oda, Y. Koya, H.Kajiyama, T. Yokoi, “Macrophage-derived extracellular vesicles regulate concanavalin A-induced hepatitis by suppressing macrophage cytokine production”, Toxicology, 2020, https://doi.org/10.1016/j.tox.2020.152544.