Ligand Internalization

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.

• Label your own ligands with pHrodo dyes
  
• See selection guide for no-wash assays

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.

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.

• Label your own ligands with Alexa Fluor dyes
  
• See selection guide for quench or wash-based assays

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.