Exosome / Endocytosis / Phagocytosis | Reagent and Kit Selection Guide

Science Note

[Oct. 15, 2024] Previous Science Note
Cancer cells hijack macrophages to metastasis

Recent research into cancer metastasis shows that cancer cells hijack immune cells, particularly macrophages, to promote metastasis. Here are some of the papers showing how macrophages contribute to cancer metastasis and immune evasion. Cancer metastasis is the spread of cancer cells from a primary tumor to distant organs, often evading immune surveillance. Immune cells, such as macrophages, can be co-opted by cancer cells to create an immunosuppressive environment that supports tumor growth and metastasis. Tumor-associated macrophages (TAMs) and other immune cells such as neutrophils and regulatory T cells can promote metastatic progression by facilitating tissue remodeling, angiogenesis and immune evasion. Conversely, enhancing the function of cytotoxic immune cells, such as CD8+ T cells and natural killer (NK) cells, may help to target and eliminate metastatic cancer cells.
Efferocytosis reprograms the tumor microenvironment to promote pancreatic cancer liver metastasis Click here for the original article: Yuliana Astuti, et. al., Nature Cancer, 2024.	Necroptosis enhances ‘don’t eat me’ signal and induces macrophage extracellular traps to promote pancreatic cancer liver metastasis Click here for the original article: Cheng-Yu Liao et. al., Nature Communications, 2024. *ECGreen (Dojindo) was used for FACS-based endocytosis analysis.	Mesothelin Secretion by Pancreatic Cancer Cells Co-opts Macrophages and Promotes Metastasis Click here for the original article: Teifion Luckett et. al., Cancer Research, 2024.
Point of Interest - Efferocytosis, the process by which macrophages engulf apoptotic cells, is physiologically used to protect host tissues from immune attack, resulting in local immunosuppression. - In pancreatic ductal adenocarcinoma (PDAC), macrophage reprogramming by efferocytosis supports pancreatic cancer liver metastasis, allowing cancer cells to evade immune detection and grow. - Macrophage progranulin regulates lysosomal acidification, which is critical for efferocytosis and macrophage conversion during metastasis. - Blocking efferocytosis or progranulin improves CD8+ T cell function and reduces liver metastasis, suggesting new therapeutic approaches.	Point of Interest - Mixed lineage kinase domain like pseudo kinase-driven necroptosis in PDAC recruits macrophages, enhances the tumor CD47 "don't eat me" signal and induces CXCL8 activation. - Necroptosis-induced CXCL8 activation initiates epithelial-mesenchymal transition and endothelial adhesion, promoting PDAC liver metastasis. - Blocking necroptosis and CD47 reduces PDAC liver metastasis, offering potential for neoadjuvant immunotherapy and radical surgery.	Point of Interest - Mesothelin secretion by PDAC cells reprograms macrophages by inducing macrophage VEGFA and S100A9 expression. - Macrophage-derived VEGFA supports tumor growth, while S100A9 promotes neutrophil infiltration and lung metastasis. - Mesothelin drives macrophage-neutrophil interactions and promotes PDAC metastasis through extracellular traps and VEGFA signaling.
Related Techniques
Apoptosis detection in multiple samples	Annexin V Apoptosis Plate Assay Kit
Phagocytosis Assay	AcidSensor Labeling Kit – Endocytic Internalization Assay and Cellstain- Calcein-AM solution
Endocytosis Detection detection	ECGreen-Endocytosis Detection
Lysosomal function	Lysosomal Acidic pH Detection Kit -Green/Red and Green/Deep Red
First-time autophagy research	Autophagic Flux Assay Kit
Plasma Membrane Staining	PlasMem Bright Green / Red
Total ROS detection	Highly sensitive DCFH-DA or Photo-oxidation Resistant DCFH-DA
Glycolysis/Oxidative phosphorylation Assay	Glycolysis/OXPHOS Assay Kit
Cell Proliferation / Cytotoxicity Assay	Cell Counting Kit-8, Cytotoxicity LDH Assay Kit-WST

Related Applications
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. 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
Accurate Measurement for Lysosomal pH changes Existing lysosomal pH detection reagents have issues with dye localization, pH sensitivity, and retention. pHLys Green is a dye that solves these issues. The improved dye retention and localization enable detection of normal lysosomes, and the improved pH sensitivity enables detection of slight pH changes.
1. High sensitive pH detection Comparison of pH response of cells treated with low concentrations of lysosomal acidification inhibitor Bafilomycin A1	2. High specificity for lysosomes Comparison of specificity for lysosomes using lysosomal marker protein LAMP1-GFP expressing cells	3. High retention in lysosomes Comparison of intracellular retention
Product in Use: - Lysosomal Acidic pH Detection Kit-Green/Deep Red Related Product: - pHLys Red- Lysosomal Acidic pH Detection

What is Plasma Membrane?

The plasma membrane (PM) separates the intracellular environment from the extracellular space and protects the cells from the extracellular environment. The PM plays important roles in selective permeation or signaling between cells etc. Observing the dynamics of the PM is useful in various research field.

Cell Membrane-related Reagents Selectable by Targets

Selesction Guide

Cell membrane

~ 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

Endocytosis/Phagocytosis

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.

Track Phagocytosis

~ Features ~

Label with pH-sensitive fluorescent dye
All-in-one Kit
Compatible with phagocytosis assay

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]
③ MT-1 MitoMP Detection Kit [code: MT13]

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

Product	Size	Product Code
Visualization of Phagocytosis	Phagocytosis Detection Dye
AcidSensor Labeling Kit	3 samples	A558-10
Note:Proteins with a molecular weight above 50,000 can be labeled.

Exosome

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)