Real-Time Analysis of AKT Signaling Activities at Single-Cell Resolution Using Cyclic Peptide-Based Probes

Here we present a protocol for interrogating AKT signaling activities in living single cells, using a pair of cyclic peptide-based fluorescent probes. These probes are encapsulated in liposomes and delivered into cells, where they continuously report on AKT signaling activities through a Föster resonance energy transfer mechanism. We describe the use of a microwell chip to achieve single-cell resolution and demonstrate the procedure for on-chip immunostaining. Finally, we provide a method for data extraction, correction, and processing.