In recent years, significant advancements have been made in the understanding of novel exo/endocytic pathways in metabolism. These discoveries have garnered considerable attention, particularly in the field of oncology. For instance, tumor-derived extracellular vesicles have been identified as critical mediators in cancer-induced hepatic reprogramming. Their role, along with TNF inhibition, offers a targetable pathway for both preventing fatty liver formation and enhancing the efficacy of chemotherapy. Another groundbreaking observation reveals that the ingestion of tumor-derived microparticles by macrophages induces a rapid metabolic and phenotypic switch. This change subsequently results in decreased motility in the early stages of metastasis in the lung. Additionally, research has demonstrated that phospholipase D6, a protein found on the mitochondrial outer membrane, accelerates the transport of LDL-LDLR from endocytic vesicles to mitochondria, thereby supporting steroidogenesis.
Tumour extracellular vesicles and particles induce liver metabolic dysfunction
Mem Dye-labeled EVs are internalized via endocytosis:
HeLa cells were incubated with 10 μmol/L ECGreen 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).
Observation of temperature-dependent endocytosis changes in floating cells
PlasMem Bright Series has low cytotoxicity, and high membrane retention of dyes and can be used in various experiments using live and fixed cells.
Clear visualization of plasma membrane
Observe morphology of neuron (differentiated SH-SY5Y cells) and localization of mitochondria in axon.
High retentivity on plasma membrane
HeLa cells stained with each plasma membrane staining reagent were incubated for 24 hrs and each the resulting fluorescent image was compared. PlasMem Bright series had higher retentivity in plasma membrane than other products.
Note: 1 tube (100 µl), 10 assays at 35 mm dish, 10 assays at μ-Slide 8 well
~ Features ~
Precise visualization of endocytosis
Track endocytosis using live cells
High responsiveness to pH change
ECGreen is a pH dependent fluorescence dye that localizes to vesicle membrane. The visualization of endocytosis using the ECGreen is a more direct method than fluorescent analogs and allows visualization endocytosis from the stage of early endosomes.
Overall, this results in increased oxidative stress and accelerated cellular damage.
Stain vesicle membrane precisely
Other companies (a fluorescent analog): intravesicular staining
Fluorescent Dye-Dextran Conjugates or membrane staining reagents are used to visualize endocytosis. However, they have limitations in observing dynamics of endosomes in live cells in terms of precision of staining or retentivity of reagent. ECGreen is the reagent that over comes the limitations.
Clear visualization of intracellular vesicular trafficking
It has been known that Wortmannin inhibits the recycling of endosomes or transition to lysosomes and causes enlargement of endosomes. To evaluate these changes caused by Wortmannin, early endosomes were co-stained by ECGreen and Rab5-RFP (marker protein of early endosomes), and lysosomes were co-stained by ECGreen and lysosome staining reagent. In adding Wortmannin, ECGreen was colocalized with enlarged endosomes (Rab5-RFP). On the other hand, ECGreen wasn’t colocalized with lysosomes.
Cover steps from fluorescence labeling to purification
Little effect on exosome properties
Recent findings suggest that exosomes, a form of extracellular vesicle (EV), 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 good target for labeling.
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).
Mem Dye-Deep Red (Purple): Ex 640 nm/Em 640-760 nm
Alternative Product “P” (Green): Ex 561 nm/Em 560-620 nm
Alternative Product “P” (Red): Ex 640 nm/Em 650-700 nm
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)
In Mem Dye-Green, Red, the aggregation of the dye was not confirmed as in Mem Dye-Deep Red.
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.
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?)
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)
Observethetime-dependent changes in exosome 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
*ExoIsolator Exosome Isolation Kit contains Filter Holder x 1, Isolation Filter x 3, Tweezers x 1. The Filter Holder can be reused after autoclaving.