Exosome / Endocytosis / Phagocytosis | Reagent and Kit Selection Guide

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.

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]
③ 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.