Flow cytometry analysis of receptor internalization/shedding.

Abstract

Differentiation, proliferation, and chemotaxis responses stem from biological programs that recognize checkpoints at the level of membrane receptor internalization and shedding. Therefore, receptor trafficking represents a crucial regulator of cell functions. Here, we present a survey of analyses of receptor internalization vs. shedding based on simple flow cytometry-based techniques. A relevant basic observation is that a fluorochrome-bearing antibody bound to a specific receptor that is translocated from the membrane to the cytoplasm continues to emit light, i.e., the cell remains equally positive for that marker even if the receptor is strongly downregulated or no longer detectable on the membrane. In contrast, fluorescence is lost following receptor shedding. The combined uses of standardized hyperosmolar sucrose, acidic treatment and flow cytometry staining at different times allows for fully informative studies of the internalization or shedding pattern of a given receptor. The procedure can be simplified into a straightforward, simple-to-use, and flexible flow cytometry method based on two sequential steps with in-between receptor stimulation. This method obviates the need for time-consuming fluorescence techniques and even confocal microscopy. We validate this procedure via comparisons of three receptors, i.e., CXCR4, CD30, and CD25, with membrane trafficking patterns that are involved in biological functions that are relevant to immunity and cancer. © 2016 International Clinical Cytometry Society.