Exosome / Endocytosis / Phagocytosis | Reagent and Kit Selection Guide

Science Note

[Jun. 26, 2024]                                                                                                                                                                                                                            Previous Science Note
New function of endocytosis induced and inhibited by membrane proteins

Endocytosis induction refers to the initiation of the endocytic process, where specific signals trigger the cell to internalize extracellular substances. This induction often involves membrane proteins that can act as receptors to recognize and bind external ligands, initiating the formation of endocytic vesicles. The interaction between ligands and membrane proteins not only triggers endocytosis, but also ensures that the internalized cargo is selectively processed. Thus, membrane proteins are integral to both the initiation and specificity of endocytosis, influencing cellular uptake and signaling pathways.

Endocytic vesicles act as vehicles for glucose uptake in response to growth factor stimulation
Click here for the original article: Ryouhei Tsutsumi, et. al., Nature Communications, 2024.

Endocytosis blocks the vesicular secretion of exosome marker proteins
Click here for the original article: Yiwei Ai et. al., Science Advances, 2024.

Cell surface protein aggregation triggers endocytosis to maintain plasma membrane proteostasis
Click here for the original article: David Paul et. al., Nature Communications, 2023.

Point of Interest
- Platelet-derived growth factor(PDGF) enhances cellular glucose uptake via receptor endocytosis and independently of known mechanisms, resulting in a near doubling of glucose uptake by the cells.

- PDGF receptor (PDGFR) co-endocytoses with subset of glucose transporter 1 (GLUT1/SLC2A1) upon PDGF-stimulation.

- The PDGFR/GLUT1-containing endosomes have multiple glycolytic enzymes and localize to adjacent mitochondria.

- The glucose-loaded endosomes generated by growth factors deliver glucose to the glycolytic machinery in proximity to mitochondria.

Point of Interest
- Endocytosis inhibition induces plasma membrane accumulation and vesicular secretion of CD63.

- High expression of CD63, CD81 or CD9 inhibits its own endocytosis and induces its plasma membrane accumulation and vesicular secretion.

- Induction of endocytosis inhibits their vesicular secretion and, in the case of CD9 and CD81, causes their destruction in the lysosome.

- Vesicular secretion of exosome marker proteins occurs primarily through an endocytosis-independent pathway.

Point of Interest
- Aggregation of protein ectodomains by cross-linking antibodies ability triggers specific and fast endocytosis of the receptor, independent of clathrin and dynamin.

- Upon aggregation, even canonical clathrin-dependent cargoes are redirected to the aggregation-dependent endocytosis (ADE) pathway.

- ADE is an actin-driven process that morphologically resembles macropinocytosis.

- ADE clears stress-induced receptor aggregates and facilitates their lysosomal degradation to maintain cell surface proteostasis.

Related Techniques
           Endocytosis Detection detection ECGreen-Endocytosis Detection
           Exosome Labeling ExoSparkler Exosome Membrane Labeling Kit-Green / Red / Deep Red
           Lysosomal function Lysosomal Acidic pH Detection Kit -Green/Red and Green/Deep Red
           Plasma Membrane Staining PlasMem Bright Green / Red
           Phagocytosis Assay AcidSensor Labeling Kit – Endocytic Internalization Assay and Cellstain- Calcein-AM solution
           Autophagy detection Autophagic Flux Assay Kit
           Mitophagy or autophagy detection Mitophagy Detection Kit
           Glycolysis/Oxidative phosphorylation Assay Glycolysis/OXPHOS Assay Kit
Related Applications

Clear visualization of intracellular vesicular trafficking

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.

Endosomes (ECGreen, green): Ex. 405 nm / Em. 500 – 560 nm
Early endosomes (Rab5-RFP, red): Ex. 561 nm / Em. 560 – 620 nm
Recycling endosome (Transferrin-Alexa fluor 488 conjugate, red: pseudo-color): Ex. 488 nm / Em. 500 – 550 nm
Late endosomes (Rab7-RFP, red): Ex. 561 nm / Em. 560 – 620 nm
Lysosomes (Lamp1-RFP, red): Ex. 561 nm / Em. 560 – 620 nm

[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

Phagocytosis assay of labeled apoptotic cells in THP-1 cells



AcidSensor-labeled substances are taken up by cells and their fluorescence increases when they reach acidic organelles such as lysosomes. Taking advantage of this property, we evaluate the phagocytic activity of apoptotic cells by co-culturing AcidSensor-labeled apoptotic cells with Calcein-labeled THP-1 macrophages.  As a result, Calcein (Green) / AcidSensor (Deep red) double-positive cells, indicating THP-1 macrophages phagocytosing apoptotic cells, were observed by flow cytometry (Fig. 1a). Furthermore, when the phagocytosis of THP-1 macrophages was inhibited by Cytochalasin D, the percentage of double-positive cells decreased (Fig. 1b and 1c), confirming that the assay system can accurately evaluate phagocytosis.

A recent report reveals that inhibition of mitochondrial function induces a switch to glycolysis and reduces phagocytosis in cultured microglia, resident macrophages in the central nervous system*. To replicate this result, phagocytosis assays were performed using mitochondria-inhibited THP-1 macrophages. The results show that FCCP, a potent uncoupler of oxidative phosphorylation in mitochondria, decreases mitochondrial membrane potential (MT-1, Red) of THP-1 macrophages (Fig. 2) and reduces phagocytosis (Fig. 3).

*Lauren H. Fairley, et al., PNAS (2023)

Products in Use
① AcidSensor Labeling Kit – Endocytic Internalization Assay [code: A558]
② -Cellstain- Calcein-AM solution [code: C396]
 MT-1 MitoMP Detection Kit [code: MT13]

[Experimental Procedure]

Preparation of AcidSensor-labeled apoptotic cells (day before assay)

1. Add 10 μl of DMSO to NH2-Reactive AcidSensor and dissolve. 5 μl NH2-Reactive AcidSensor solution was added to 5 ml HBSS to make the Working solution (1000-fold dilution).
2. Wash Jurkat cells twice with HBSS.
3. Jurkat cells (5×107 cells) were transferred to the tube and the supernatant was removed after centrifugation.
4. Add the Working solution to the tube with Jurkat cells (5×107 cells) and suspend.
5. Incubate at 37°C for 30 minutes for labeling with AcidSensor.
6. After labeling, the cells were washed twice with HBSS.
7. AcidSensor-labeled Jurkat cells were suspended in RPMI medium with 10% FBS added with 0.5 μM staurosporine.
8. Apoptosis was induced by o/n culture in an incubator (37°C, 5% CO2) for 18 hours.
9. After induction of apoptosis, the cells were washed with culture medium and used for the phagocytosis assay.


Phagocytosis assay using THP-1 macrophages

1. To differentiate THP-1 cells into macrophages, THP-1 cells were seeded into 6 well plates at 1x106 cells/well and incubated with 100 nM PMA for 3 days in an incubator.
2. After washing THP-1 macrophages twice with HBSS, Calcein-AM (Code: C396, 0.5 μg/ml) and MT-1 (Code: MT13, 1000-fold dilution) included HBSS solution was added to the wells and incubated in the incubator for 30 minutes.
3. Wash twice with a culture medium.
4. The cells were incubated with 10 μM Cytochalasin D for 1 hour or 5 μM FCCP for 30 minutes in the incubator.
5. After twice washing with culture medium, AcidSensor-labeled apoptotic cells (3x106 cells/well) were added to the well with or without Cytochalasin D or FCCP. The cells were incubated in the incubator for 4 hours.
6. Wash twice with HBSS.
7. Add 500 μl Imaging buffer solution (Code: MT13) and collect THP-1 macrophages from plates with a cell scraper.
8.Cell suspensions were analyzed by flow cytometry.



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