Single Cell Fluorescence Ratio Image Analysis for Studying ESCRT Function in Receptor Trafficking.

The endosomal sorting complexes required for transport (ESCRT) comprise a major trafficking pathway for plasma membrane proteins and are fundamental for ubiquitin-dependent cargo endocytosis. Here, we describe a method for studying the effect of ESCRT complexes on endo-lysosomal membrane trafficking and their role in receptor integrin α5β1 downregulation. Single cell fluorescence ratio image analysis (FRIA), using appropriate fluorescence probes, enables the measurement of dynamics of integrin α5β1 containing vesicles and represents a live cell-based method for studying the role of ESCRTs.