UNIT 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging
Published Online: 1 SEP 2008
Copyright © 2008 by John Wiley & Sons, Inc.
Lab Protocol Title
Current Protocols in Cell Biology
How to Cite
Barriere, H. and Lukacs, G. L. 2008. Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging. Current Protocols in Cell Biology. 40:15.13.1–15.13.21.
- Published Online: 1 SEP 2008
- Published Print: SEP 2008
The post-endocytic sorting of internalized membrane proteins plays a critical role in numerous physiological processes, including receptor desensitization, degradation of non-native plasma membrane proteins, and cell surface retrieval of receptors from early endosomes upon ligand dissociation. Here, we describe a fluorescence ratiometric image analysis (FRIA) method used to determine the post-endocytic fate and transport kinetics of transmembrane proteins based on the pH measurement of internalized cargo-containing compartments in living cells. The method relies on the notion that the pH of a cargo-containing transport vesicle (vesicular pH, pHv) could be taken as an indicator of its identity, considering that endocytic organelles (e.g., sorting endosome, recycling endosome, late endosome/MVB, and lysosome) have characteristic pHv. The pH-sensitive FITC-conjugated secondary antibody is attached to the cargo via a primary antibody, recognizing the cargo extracellular domain. The pHv is determined by single-cell FRIA. Internalized cargo colocalization with organellar markers, as well as pHv measurement of recycling endosome, lysosome, and the TGN are discussed to validate the technique and facilitate data interpretation. Curr. Protoc. Cell Biol. 40:15.13.1-15.13.21. © 2008 by John Wiley & Sons, Inc.
- post-endocytotic sorting;
- fluorescence ratiometric image analysis;
- endocytic organelles