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)
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.
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.
Figure 3. Wortmannin-induced inhibition the recycling of endosomes or transition to lysosomes
Excitation and emission spectra of ECGreen
λex: 386 nm
λem: 522 nm
<Recommended filter settings>
Ex: 350 – 410 nm, Em: 500 – 560 nm