外泌体是细胞外囊泡 (EV) 的一种形式，可能导致癌症的恶性转化和转移。 因此，通过外泌体进行的细胞间通讯引起了科学界的极大兴趣。 为了阐明这种交流，已经使用了基于荧光染料的标记技术。 标记细胞膜的荧光染料通常用于外泌体标记，因为外泌体中的脂质双层是合适的标记目标。 ExoSparkler 系列可用于对纯化的外泌体膜或蛋白质进行染色，从而可以对细胞摄取的标记外泌体进行成像。
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.1,2The 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).
Experimental conditionsExosomes 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.Detection conditionsMem Dye-Deep Red(Purple): Ex 640nm/Em 640-760nmAlternative Product “P” (Green): Ex 561nm/Em 560-620nmAlternative 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 propertiesNTA (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 exosomesExosomes 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 exosomesExosomes 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 purificationExoSparkler 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
<Experimental conditions>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.
<Detection Conditions>Mem Dye-Deep Red (Purple): Ex 640 nm/Em 640-760 nmLysosome 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
Experimental conditionsExosomes 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.Detection conditionsGreen: Ex 488nm/Em 490-540nmRed: Ex 561nm/Em 570-640nmDeep Red: Ex 640nm/Em 640-760nm
Unlabeled dye is likely to be retained inside the filter, but we have confirmed that the unlabeled dye is not remaining on top of the filter. Please refer to the following experiment:<Experimental condition>① We stained the 10 µg exosomes (as protein amount) purified by ultracentrifugation.② We also prepared only using buffer as a control3. The recovered solution is added to HeLa cells (1.25 x 104 cells), and the fluorescence image was observed 4 hours later.As a result, it was confirmed that no bright fluorescent particles (unlabled dyes) were observed in the cells using the recovered solution with the only buffer.① Exosome + Buffer
② Only Buffer
Fluorescent images at 4 h incubation
Detection conditionsGreen：Ex 488 nm / Em 490 – 540 nmRed ：Ex 561 nm / Em 570 – 640 nmDeep Red：Ex 640 nm / Em 640 – 760 nm
We can not recommend to store the exosomes after it has been stained.Please use the stained exosomes as soon as possible.