Endosomal proteins play a critical role in determining cell fate by regulating the trafficking and signaling of receptors and other membrane-bound molecules within cells. These proteins control the endocytosis, recycling, or degradation of signaling receptors, thereby influencing key pathways such as those governing differentiation, survival, and apoptosis. By modulating the duration and intensity of signaling cascades, endosomal proteins help determine whether a cell will proliferate, differentiate, or undergo programmed cell death. Dysregulation of these processes can lead to inappropriate cell fate decisions and contribute to diseases such as cancer and neurodegeneration.
MYCT1 controls environmental sensing in human haematopoietic stem cells
Click here for the original article: Júlia Aguadé-Gorgorió et. al., Nature, 2024. *ECGreen (Dojindo) was used for FACS-based endocytosis analysis.
EGFR-dependent endocytosis of Wnt9a and Fzd9b promotes β-catenin signaling during hematopoietic stem cell development in zebrafish
Click here for the original article: Nicole Nguyen et. al., Science Signaling, 2024.
Point of Interest
-Ku70 deficiency increases susceptibility to colon cancer by disrupting DNA repair and activating aberrant signaling pathways.
-Ku70 forms a complex with GTPase Ras and kinase Raf at Rab5+Rab7+ early-late endosomes, activating MEK-ERK pathways that reduce cell proliferation and tumorigenesis.
-Targeting this Ku70 signalosome could improve cancer treatment by restoring proper DNA repair and cell cycle regulation.
Point of Interest
-MYCT1 is a key regulator of human hematopoietic stem cell (HSC) self-renewal and expansion by modulating endocytosis and environmental sensing.
-MYCT1 expression is essential for HSC expansion and engraftment, and its loss leads to excessive endocytosis and dysregulated signaling.
-Restoring MYCT1 in cultured HSCs preserves stemness and improves expansion, highlighting its importance in human HSC regulation.
Point of Interest
-EGFR and EPS15-dependent Wnt9a-Fzd9b signaling is essential for hematopoietic stem cell development in zebrafish.
-EGFR-mediated phosphorylation triggers endocytosis of Wnt9a-Fzd9b complexes, which are necessary for hematopoietic stem cell signaling.
-EPS15 promotes Wnt9a-Fzd9b endocytosis, which is critical for hematopoietic stem cell development in zebrafish and humans.
Phagocytosis assay of labeled apoptotic cells in THP-1 cells
Wortmannin is known to inhibit endosomal recycling and lysosomal translocation, leading to endosomal enlargement.
These changes induced by Wortmannin were confirmed by co-staining with ECGreen (green) and the following indicators.
①Eary endosome: Rab5-RFP (red)
② Recycling endosome: Fluorescent labeled Transferin (red)
③ Late endosome: Rab5-RFP (red)
④ Lysosome: Lamp1-RFP (red)
As a result, it was confirmed that ECGreen (green) co-localizes only with enlarged early endosomes and recycling endosomes (Fig. ① and ②), but not with late endosomes or Lysosomes (Fig. ③ and ④), supporting Wortmannin's effect. ECGreen can visualize changes in the intracellular vesicular trafficking system and endosome shape.
[Experimental Procedure]
(1) Prepare HeLa cells in 8 wells of μ-Slide and incubate overnight.
(2) After washing with HBSS, 200 µl of Wortmannin (final concentration: 100 nmol/l) prepared in 10% FBS-containing MEM medium was added.
(3) Incubate at 37°C for 30 minutes
(4) 200 µl of ECGreen (diluted 1,000-fold) prepared in 10% FBS-containing MEM medium without removing the supernatant
(5) Incubate at 37°C for 30 minutes
(6) Wash the cells twice with HBSS and add MEM medium containing 10% FBS.
(7) Observation with a confocal laser microscope
Selesction Guide
~ Features ~
Applicable to live and fixed cells
High retentivity of reagents with low toxicity
Just add reagents into medium
Low toxicity, No washing, and High retentivity
Comparison with other products
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
Track endosome
~ 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.
Detection Conditions
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.
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