Analysis of endocytic trafficking by single-cell fluorescence ratio imaging.

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, pH(v)) 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 pH(v). The pH-sensitive FITC-conjugated secondary antibody is attached to the cargo via a primary antibody, recognizing the cargo extracellular domain. The pH(v) is determined by single-cell FRIA. Internalized cargo colocalization with organellar markers, as well as pH(v) measurement of recycling endosome, lysosome, and the TGN are discussed to validate the technique and facilitate data interpretation. (c) 2008 by John Wiley & Sons, Inc.