Ligand internalization is a receptor-mediated endocytic process in which the cell will only take in an extracellular molecule if it binds to its specific receptor protein on the cell’s surface. It is an important cell signaling event, and Life Technologies offers a variety of fluorescent ligands targeted to common receptors.

 

 

 


No-wash assays

Specific receptor-mediated endocytosis pathways can be investigated with fluorescent ligands including pHrodo™ dyes targeted to common receptors, including, EGF, LDL, and transferrin.

The most convenient, no-wash assay format uses pHrodo™ Red or pHrodo™ Green to allow discrimination of stages in the endocytic pathway from early endosome to lysosome formation with no quench or wash required.

pHrodo™ dyes are essentially non-fluorescent at neutral pH and exhibit increasing signal with a red or green readout respectively as the pH decreases. The increase in fluorescent signal can be used to monitor progression in the endocytic pathway.

Ligand Internalization

Mouse monocyte/macrophage cells (MMM cells, ATCC) treated with transferrin conjugated with pHrodo™ Red in Live Cell Imaging Solution and counterstained with NucBlue® Live ReadyProbes™ Reagent. Punctate red spots are visible where labeled transferrin was internalized.


Quench or wash-based assays

Specific receptor-mediated endocytosis pathways can be investigated with a variety of fluorescent ligands targeted to common receptors, including, EGF, LDL, and transferrin.

Alexa Fluor® dyes conjugated to a range of ligands provide a choice of wavelength options and single-emission measurement for each. Assays require a wash step to remove unbound conjugates from the cell surface or quenching of the extracellular signal.

Ligand Internalization

Visualization of transferrin and transferrin receptors in A431 cells.

A431 cells incubated with green-fluorescent Alexa Fluor® 488 transferrin, then fixed and permeabilized. Transferrin receptors were identified with anti–transferrin receptor, mouse IgG1 monoclonal antibody and visualized with red-fluorescent Alexa Fluor® 555 goat anti–mouse IgG antibody. Yellow fluorescence indicates regions of co-localization. Nuclei were stained with DAPI.

Ligand internalization selection guides

 
Readout
No-wash assay to image receptor-mediated endocytosis by fluorescence intensity
Range
Monitors early endosome to early lysosome formation
Vehicle
EGF 
Transferrin 
Common filter set
TRITC
FITC
TRITC
Labels
pHrodo™ Red
pHrodo™ Green
pHrodo™ Red
Ex/Em (nm)
560/585
509/533
560/585
Signal-to-noise ratio
Photostability
Multiplexing
Yes
Live cells
Yes
Fixed cells
No
Fixable
Yes
Platforms*
I, M, FC
Format
20 µg
20 µg
1 mg
Cat. No.
* I = Imaging, M = Microplate, FC = Flow cytometry.
 
Readout
Quench or wash-based assay to image receptor-mediated endocytosis by fluorescence intensity
Range
Fluorescent signal is not modulated by endocytic progress
Vehicle
LDL
Transferrin
EGF
Common filter set
Texas Red®
FITC
FITC
Texas Red®
FITC
TRITC
Labels
Alexa Fluor® 594
Alexa Fluor® 488
Alexa Fluor® 488
Alexa Fluor® 594
Alexa Fluor® 488
Alexa Fluor® 555
Ex/Em (nm)
590/617
495/519
495/519
590/617
495/519
555/565
Signal-to-noise ratio
Photostability
Bibliography
Multiplexing
Yes
Live cells
Yes
Fixed cells
No
Fixable
Yes
Platforms*
Imaging
Format
200 µL
200 µL
5 mg
5 mg
100 µg
100 µg
Cat. No.