FRET Imaging by Laser Scanning Cytometry on Large Populations of Adherent Cells.

FRET Imaging by Laser Scanning Cytometry on Large Populations of Adherent Cells. Curr Protoc Cytom. 2014;70:2.23.1-2.23.29 Authors: Doan-Xuan QM, Szalóki N, Tóth K, Szöllősi J, Bacso Z, Vámosi G Abstract The application of FRET (fluorescence resonance energy transfer) sensors for monitoring protein-protein interactions under vital conditions is attracting increasing attention in molecular and cell biology. Laser-scanning cytometry (LSC), a slide-based sister procedure to flow cytometry, provides an opportunity to analyze large populations of adherent cells or 2-D solid tissues in their undisturbed physiological settings. Here we provide an LSC-based three-laser protocol for high-throughput ratiometric FRET measurements utilizing cyan and yellow fluorescent proteins as a FRET pair. Membrane labeling with Cy5 dye is used for cell identification and contouring. Pixel-by-pixel and single-cell FRET efficiencies are calculated to estimate the extent of the molecular interactions and their distribution in the cell populations examined. We also present a non-high-throughput donor photobleaching FRET application, for obtaining the required instrument parameters for ratiometric FRET. In the biological model presented, HeLa cells are transfected with the ECFP- or EYFP-tagged Fos and Jun nuclear proteins, which heterodimerize to form active AP1 transcription factor. Curr. Protoc. Cytom. 70:2.23.1-2.23.29. © 2014 by John Wiley & Sons, I...
Source: Current Protocols in Cytometry - Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research