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.