Applications of FRET in Flow Cytometry

Gary Warnes and Sandra Martins

Flow Cytometry Core Facility, Institute of Cell and Molecular Science, Barts and The Royal London Schools of Medicine and Dentistry, London University, 4, Newark Street, London, E1 2AT, UK.

Fluorescence Resonance Energy Transfer (FRET) is characterized by the non-radiative transfer of energy between donor and acceptor fluorophores after excitation of the donor by a light source. This occurs via a long range dipole-dipole coupling mechanism when molecules are in a parallel orientation and within 10 nm. Flow Cytometric use of FRET has become more prevalent due to increased use of Fluorescent Protein (FP) technology to investigate not only molecule proximity but protein functionality when single molecules are dual labelled with two types of FP e.g. CFP and YFP. Here we describe how a BD FACSAria cell sorter and LSRII were used to measure CFP-YFP FRET, reporting activity of CCF2 substrate and BrdU detection by PI-ToPro-3 FRET. YFP was collected in 530/25nm argon channel, CFP was collected in 450/40nm violet channel and FRET was collected in the 530/25nm violet channel. MHC Class I peptide processing time in the Endoplasmic Reticulum (ER) can be measured by flow cytometric FRET after tagging of MHC class I with YFP and the protein editor, Tapasin with CFP. The biological activity of single proteins can also be studied by FRET, the protein Cdc42 is a member of the Rho family G proteins and triggers filopodium formation during cell movement. Expression of Cdc42 with CFP and YFP at the c and n terminus of the protein allows a biological read out of the functionality of this protein in the presence or absence of Cdc42 inhibitors by flow cytometric analysis of FRET. FRET Ratios were calculated via ratioing of median fluorescence values of the FRET channel from the FRET sample and CFP control (with background correction). Other uses of FRET include the use of the gene reporting substrates CCF2 and CCF4 as well as BrdU detection by the use of PI and ToPro3. Cell sorting can be an aid to the imaging of FRET by maintaining cell cultures that have a high proportion of cells displaying FRET.