Measuring ERK Activity Dynamics in Single Living Cells Using FRET Biosensors
Fluorescence resonance energy transfer (FRET)-based biosensors are powerful tools for measuring spatio-temporal signaling dynamics in single living cells with subcellular resolution. There are quite a number of already existing sensors and this technology is increasingly used to obtain quantitative dynamic datasets. In this chapter, we describe the analysis of endogenous extracellular signal-regulated kinase (ERK) activity in living cells using the EKAR2G (ERK activity reporter second generation) probe. We focus on the generation of stable cell lines expressing the EKAR2G sensor as well as data acquisition and analysis.
Key wordsFluorescence resonance energy transfer (FRET) Ratiometric FRET imaging Biosensors EKAR2G ERK dynamics Cell-cell variability Signaling heterogeneity Cell fate Live-cell microscopy High throughput PC12 cells
This work was supported by grants from the Human Frontier Science Program, the Swiss National Science Foundation, and the Novartis Foundation for medical-biological research.