Background

My project is focused on the development of a new multiplexed assay that also relies on flow cytometry for HTS. We became interested in the idea of choosing which specific intracellular targets we want to screen for as opposed to relying on what is found naturally. For this reason, we decided to exploit the diversity of FRET based sensors that have been created in the development of our assay.

Forster Resonance Energy Transfer (FRET) is the process of energy transfer from a donor fluorophore to an acceptor fluorophore. It is a distant dependent process and relies on a spectral overlap of excitation and emission of acceptor and donor, respectively. The FRET sensors used in our method are intramolecular; the donor (CFP) and acceptor (YFP) are bound to the same protein. When the protein undergoes a conformational change upon activation, the fluorophores become closer or further from one another, changing the amount of FRET occurring. This FRET is calculated as YFP emission/CFP emission.

Flow cytometry allows multiple reporters to be measured at once even if the reporters are not spectrally distinct by labeling the cells in a spare channel. Therefore, by using cellular dyes, we can barcode our cells expressing different FRET sensors and be able to tell each sensor apart in a mixed sample.  

Method

FRET sensors for different intracellular targets are stably expressed in cells. The cells are loaded with different concentrations cellular dyes. The different loading conditions of the cells correspond to a sensor, allowing us to tell the sensors apart. The loaded cells are then mixed together and stimulated. The mixed population of cells is read on the FACS Aria.