Quantifying cellular internalization with a fluorescent click sensor

The ability to determine the amount of material endocytosed by a cell is important for our understanding of cell biology and in the design of effective carriers for drug delivery. To quantify internalization by fluorescence, the signal from material remaining on the cell surface must be differentiated from endocytosed material. Sensors for internalization offer advantages over traditional methods for achieving this as they exhibit improved sensitivity, allow for multiple fluorescent markers to be used simultaneously, and are amenable to high-throughput analysis. We have developed a small fluorescent internalization sensor, similar in size to a standard fluorescent dye, that can be conjugated to proteins and uses the rapid and highly specific bio-orthogonal reaction between a tetrazine and a trans-cyclooctene group to switch off the surface signal. The sensor can be attached to a variety of materials using simple chemistry and is compatible with flow cytometry and fluorescence microscopy, making it a useful tool to study the uptake of material into cells.