Product Description
Endocytosis is a cellular process that involves macromolecules being taken up through a plasma membrane-derived vesicle called an endosome. The endocytic pathway contributes to the maintenance of intracellular homeostasis by bringing in various nutrients to the cell and transporting unwanted components to the lysosome, which acts as a waste disposal system. Recent findings reveal that disruption of endocytosis is related to certain neurodegenerative disorders and immune diseases. Consequently, investigation of the endocytic pathway is attracting considerable interest in the scientific community.
The general number of usable assays per 40 ul
– 35mm dish x 20
– μ-Slide 8 well x 20
ECGreen-Endocytosis Detection 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.
Figure 1. The detection mechanism of endocytosis
Observation of endocytosis inhibition by low temperature incubation in HeLa cells
HeLa cells were stained with ECGreen. Endocytosis was then inhibition by low temperature .:
Figure 2. The effect of low temperature incubation on endocytosis
A : Normal condition (37°C incubation) , B : Endocytosis inhibition (4°C incubation)
Detection condition:
ECGreen filter sets: 405 nm (Ex), 500 – 560 nm (Em)
Scale bar: 10 μm
Stain vesicle membrane precisely
Fluorescent Dye-Dextran Conjugates or membrane staining reagents are used to visualize endocytosis. However, they have limitations in observing the dynamics of endosomes in live cells in terms of precision of staining or retentivity of reagent. ECGreen is the reagent that over comes these limitations.
ECGreen-Endocytosis Detection 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.
ECGreen-Endocytosis Detection is more sensitive to pH changes than exiting fluorescently labeled dextrans.
Therefore, ECGreen-Endocytosis Detection is appreciable for detection of early endosomes with high sensitivity.
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.
<Detection conditions>
Endosomes (ECGreen, green): Ex. 405 nm / Em. 500 - 560 nm
Early endosomes (Rab5-RFP, red): Ex. 561 nm / Em. 560 - 620 nm
Late endosomes (Rab7-RFP, red): Ex. 561 nm / Em. 560 - 620 nm
Lysosomes (Lamp1-RFP, red): Ex. 561 nm / Em. 560 - 620 nm
<Protocol>
(1) Preapare 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
Experimental example: Observation of temperature-dependent changes in endocytosis using suspension cells
The temperature-dependent changes in endocytosis of Jurkat cells were visualized using ECGreen-Endocytosis Detection (product code: E296) and PlasMem Bright Red.
<Detection Condition>
Endosomes (ECGreen, green): Ex. 405 nm / Em. 500 - 560 nm
Plasma membrane (PlasMem Bright Red, red): Ex. 561 nm / Em. 560 - 700 nm
<Protocol>
(1) Add Jurkat cell suspension (10% FBS, RPMI) in a sample tube and incubate at 4
oC or 37
oC for 30 min.
(2) Dilute the ECGreen solution 1,000-fold with the suspension from (1).
(3) Incubate at 4
oC or 37
oC for 30 min.
(4) Wash the cells twice with HBSS.
(5) Add medium containing PlasMem Bright Red (100-fold dilution) and suspend the cells.
(6) Transfer the suspension to an imaging plate and observe the cells under a confocal microscope.
Experimental example: Time-lapse imaging of Endosome localization
<Detection conditions>
Endosomes (ECGreen, green): Ex. 405 nm / Em. 500 - 560 nm
Lysosome (Lysosome staining dye, red): Ex. 561 nm / Em. 600 - 700 nm
<Protocol>
(1) HeLa cells and culture them overnight.
(2) Wash the cells once with HBSS.
(3) ECGreen (diluted 1,000-fold) prepared in 10% FBS-containing MEM medium and lysosomal staining dye (final concentration: 100 nmol/l) were added.
(4) Observe unwashed cells at each hour using a confocal laser microscope.
Visualization of EVs uptake via endocytic pathway
Mem Dye-labeled EVs are internalized via endocytosis:
HeLa cells were incubated with 10 μmol/L ECGreen for 30 min. Then, Mem Dye-Deep Red (
Code: EX03) labeled EVs (quantified as 10 µg of protein) were added to HeLa cells. After 30 or 120 min incubation, the cells were washed and observed under a fluorescence microscope (Scale Bar: 10 µm).