Quantitative analysis of transferrin cycling by automated fluorescence microscopy

Surface receptors are transported between the plasma membrane and intracellular compartments by various endocytic mechanisms and by recycling via different pathways from sorting or recycling endosomes. The analysis of cellular components involved in mediating or regulating these transport steps is of high current interest and requires quantitative methods to determine rates of endocytosis and/or recycling. Various biochemical procedures to measure uptake of labeled ligand molecules or internalization and reappearance of surface-labeled receptors have been developed. Here, we describe a quantitative method based on fluorescence microscopy of adherent cells taking advantage of the transferrin (Tf) receptor as the prototype of cycling transport receptors. Tf is endocytosed with bound Fe(3+) and, upon release of the iron ion in endosomes, recycled as apo-Tf together with the receptor. To follow the ligand-receptor complex, fluorescently labeled Tf is used and detected microscopically with or without releasing Tf from cell surface receptors by acid stripping. To go beyond the observation of a few individual cells, automated fluorescence microscopy is employed to image thousands of cells at different time points and in parallel with different treatments (such as chemical inhibitors, siRNA silencing, or transfection of candidate genes) in a 96-well format. Computer-assisted image analysis allows unbiased quantitation of Tf content of each cell and to distinguish between different cell populations.