Pros and Cons of FRET measurement with LUB08, a new long-shift probe

Forster’s Resonance Energy Transfer (FRET) takes place between donor and acceptor fluorophores at 1-10nm proximity and thus can be used to assess molecular interactions upon proper (e.g. immunofluorescent) labeling of the molecules of interest. A typical approach for measuring FRET, especially in flow cytometry, is to measure the signal intensities in the donor, acceptor and FRET channels, and calculating FRET efficiency using spectral spillover factors. While overlap of donor emission with acceptor excitation is requirement for FRET, the other overlaps which are collateral with this decrease the proportion of the useful FRET signal. One possible way to preserve the overlap integral but reduce other spillovers is the use of dyes that have a long Stokes-shift.
The aim of work was to investigate a new such dye, LUB08, for its suitability as a FRET donor.
We have obtained absorption and emission spectra, conjugated the dye to antibodies and compared FRET obtained with LUB08 as the donor and Alexa Fluor 647 as the acceptor to that with Alexa Fluor 546 as the donor. Measurements were done with a FACSAria flow cytometer, after labeling two epitopes of the highly expressed ErbB2 molecule on the surface of SK-BR-3 cells with monoclonal antibodies Trastuzumab (TR) and Pertuzumab (PR). The donor and acceptor epitopes were also swapped for control purposes. Cell-by-cell FRET was evaluated using the program FCS express.
LUB08 was excited reasonably well by the 488 nm Ar ion laserline (ε=41360) and has shown a large Stokes-shift of ~120nm. The LUB08/AF647 pair gave stable, reproducible FRET results with LUB08 both on TR and PR, and comparable to the AF546-TR/AF647-PR pair. As expected, the acceptor signal was not influenced by LUB08 fluorescence. However, the FRET signal was contaminated with LUB08 signal, and the cell-by-cell FRET histograms were more dispersed than in the case of the conventional FRET pair.
Overall, LUB08 is potentially a useful FRET donor, but for flow cytometry it needs an acceptor which is spectrally better fitted than AF647. For a microscopic acceptor photobleaching, however, it would be ideally suited, given the lack of acceptor crosstalk into the donor signal which this method relies on.