Abstract
The following endocytosis assay has been optimized to assess EGF-stimulated EGFR endocytosis; but could be modified to assess other ligand-stimulated endocytosis of plasma membrane receptors (for which fluorochrome-conjugated ligands are available to track their receptor internalization). In brief, cells are treated with fluorescent EGF at 4 °C to allow binding to the receptor, but not internalization; then, endocytosis is allowed at 37 °C for different timepoints. For the setting up of this protocol we are really indebted to Dr. Letizia Lanzetti (Lanzetti et al., 2000).
Keywords: Internalization, Signaling, Egfr, Mammalian cells, Fluorescence
Materials and Reagents
Equipment
Software
Procedure
Day 1 Seed the appropriate number of cells (see Note 2) on top of glass coverslips placed (one per well) in 24-well cell culture dishes. Alternatively, if it is required to transfect cells prior to performing the endocytosis assay, seed cells on top of 5-6 coverslips placed in each well of 6-well culture dishes. You should prepare at least one independent multi-well dish per each time point in your experiment, plus two for controls (e.g. four dishes for an experiment with control cells plus two different times of endocytosis). Day 2 (optional) Cell DNA-transfection with Lipofectamine2000, according to manufacturer’s protocol. On the day of the endocytosis assay, transfer the coverslips with cells into 24-well cell culture dishes. Transfection could be needed when you need to modulate proteins putatively involved in the internalization of the receptor. Day 3-4 Endocytosis assay (or Day 2-3, if no cell transfection needed prior to the assay), by the following steps (see Figure 1 for the protocol scheme). Figure 1. Schematic representation of experimental flow
Notes
Recipes
Acknowledgments
This protocol was initially developed for the study Rizzolio et al. (2012). Previous reports analyzing receptors internalization that have been used as reference to set up this procedure include: Lanzetti et al. (2000); Sawamiphak et al. (2010) and Popovic et al. (2012). Correlated research activity in the authors’ lab was funded by the University of Torino-Compagnia di San Paolo, Grant ORTO11RKTW (RETHE). The authors are indebted to Dr. Letizia Lanzetti (University of Torino) for her expert support in setting up this protocol.
References
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