Quantifying EGFR endosomal recycling via immunofluorescence in breast cancer cells

Previously published protocols for quantification of endosomal recycling are limited by the use of radioactive reagents, washing of cells in reducing buffers, or the requirement for large numbers of cells. Here, we describe a protocol for quantification of endosomal recycling using immunofluorescence that is optimized for EGFR in BT-549 breast cancer cells but could be applied to other RTKs and cell lines. Our protocol enables quick assessment of recycling and uses a relatively small number of cells. For complete details on the use and execution of this protocol, please refer to Lonic et al. (2021). [Display omitted] •An optimized protocol for analysis of EGFR endosomal recycling via immunofluorescence•Used to analyze endosomal recycling in triple negative breast cancer cell line BT-549•Can be used to analyze recycling of other receptor tyrosine kinases•Allows assessment of alterations in endosomal recycling with small numbers of cells Publisher's note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Previously published protocols for quantification of endosomal recycling are limited by the use of radioactive reagents, washing of cells in reducing buffers, or the requirement for large numbers of cells. Here, we describe a protocol for quantification of endosomal recycling using immunofluorescence that is optimized for EGFR in BT-549 breast cancer cells but could be applied to other RTKs and cell lines. Our protocol enables quick assessment of recycling and uses a relatively small number of cells.