Exosome / Endocytosis | Reagent and Kit Selection Guide

Hot Topic

Extracellular vesicle-mediated communications in disease

Exosomes, a form of extracellular vesicles (EVs), mediate intercellular communication by exchanging proteins, DNA, RNA, and lipids between donor and recipient cells, and modulate the biological activities in target cells through receptor-ligand interactions. Recent findings suggest that EV-mediated communications contribute to malignant transformation and the metastasis of cancer, and several disease progressions. Consequently, EVs are attracting considerable interest in the scientific community. Today, we introduce you to three highlighted articles focusing on EVs-mediated communications related to diabetes, immunosystem, and aging.
Communication through EVs between organs in diabetes Immumoprotective effect of EVs in T cells Young EVs treatment can promote healthy aging
Extracellular vesicles mediate the communication of adipose tissue with brain and promote cognitive impairment associated with insulin resistance
(Jin Wang, et al., Cell Metabolism, 34, 1264-1279, 2022)
An intercellular transfer of telomeres rescues T cells from senescence and promotes long-term immunological memory
(Alessio Lanna, et al., Nature Cell Biology, 24, 1461-1474, 2022)
Small extracellular vesicles from young adipose-derived stem cells prevent frailty, improve health span, and decrease epigenetic age in old mice
(Jorge Sanz-Ros, et al., Science Advances, 8, eabq22, 2022)
  • - EVs and their cargo miRNAs mediate adipose tissue-brain inter-organ communication are used in this study
  • - Adipose tissue-derived EVs induce cognitive impairment associated with insulin resistance
  • - miR-9-3p in adipose tissue-derived EVs mediates synaptic damage
  • - Targeting adipose tissue-derived EVs or miRNAs prevents cognitive impairment in diabetes
  • - The common view is that T lymphocytes activate telomerase to delay senescence
  • - Upon contact with T cells, antigen-presenting cells (APCs) degraded shelterin to donate telomeres and then transferred in EVs at the immunological synapse
  • - Antigen-specific populations of T cells decide aging fate, and it is based on telomere vesicle transfer upon initial contact with APCs
  • - These telomere-acquiring T cells are protected from senescence, conferring long-lasting immune protection
  • - Small EVs (sEVs) derived from adipose mesenchymal stem cells (ADSCs) of young animals brought an improvement in several parameters usually altered with aging
  • - ADSC-sEVs induced pro-regenerative effects and a decrease in oxidative stress, inflammation, and senescence markers in muscle and kidney
  • - Predicted epigenetic age was lower in tissues of old mice treated with ADSC-sEVs and their metabolome changed to a youth-like pattern
  • - The microRNAs contained in sEVs might be responsible for the observed effects
Related Technique in This Topic
           EVs labeling ExoSparkler Exosome Membrane Labeling Kit-GreenRedDeep Red 
           EVs Isolation ExoIsolator Exosome Isolation Kit HOT
           Endocytosis detection ECGreen
           Endocytic internalization assay AcidSensor Labeling Kit HOT
           Lysosomal function assay Lysosomal pH and mass detection Kit HOT
           Autophagy detection DAPGreen / DAPRed (Autophagosome detection), DALGreen (Autolysosome detection)
           Cellular senescence detection SPiDER-βGal
           Glucose assay Glucose Assay Kit-WST
           Lipid droplets detection Lipi-Blue / Green / Red / Deep Red

 


Learn more about application data with multiple products here

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.


Product Size Product Code
Visualization of Cell membrane Plasma Membrane Staining Dyes
PlasMem Bright Green 100 µl x 1 P504-10
PlasMem Bright Red 100 µl x 1 P505-10

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.


Product Size Product Code
Visualization of Endocytosis Endocytosis Detection Dye
ECGreen-Endocytosis Detection 40 µl x 1 E296-10

Note:1 tube (40 µl), 20 assays at 35 mm dish, 20 assays at μ-Slide 8 well

Exosome Membrane/Protein Fluorescence Labeling Kit

~ Features ~

  • Not allow extracellular aggregation
  • 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


Product Size Product Code
Visualization of Exosome Exosome Membrane Fluorescence Labeling Kits
ExoSparkler Exosome Membrane Labeling Kit-Green 5 samples EX01-10
ExoSparkler Exosome Membrane Labeling Kit-Red 5 samples EX02-10
ExExoSparkler Exosome Membrane Labeling Kit-Deep Red 5 samples EX03-10
  Exosome Protein Fluorescence Labeling Kits
ExoSparkler Exosome Protein Labeling Kit-Green 5 samples EX04-10
ExoSparkler Exosome Protein Labeling Kit-Red 5 samples EX05-10
ExoSparkler Exosome Protein Labeling Kit-Deep Red 5 samples EX06-10

Note:  Protein amount : 1-10μg/ sample, Particle count : 10 to 100 x 108 /samples (As purified exosome using ultracentrifugation)

ExoIsolator Exosome Isolation Kit

~ Features ~

  • Easy to use, no technique required
  • Recovery rate equivalent to ultracentrifugation
  • Filter holder is reusable

Easy to use no technique required

Recovery rate equivalent to ultracentrifugation

(a) Nanoparticle number and (b) expression level of exosome markers

 


Product Size Product Code
ExoIsolator Exosome Isolation Kit 3 tests EX10-10
Filter for exosome isolation kit 10 pieces EX11-10

 

*ExoIsolator Exosome Isolation Kit contains Filter Holder x 1, Isolation Filter x 3, Tweezers x 1. The Filter Holder can be reused after autoclaving.

 


Product Classification

Product Classification