Using Flow Cytometry for Fluorescence Resonance Energy Transfer
A marriage of sorts
Fluorescence resonance energy transfer, or FRET, is often done using a microscope, which means it can be difficult to analyze large numbers of cells in one sitting. One way to overcome this, is by combining FRET with fluorescent-activated cell sorting (FACS), giving you a high-throughput method to screen for protein interactions in a vast array of cells.
For those of you not familiar with FRET, here is some background info:
FRET: The easy version
FRET was originally developed by the German scientist, Theodore Förster, and is sometimes called Förster resonance energy transfer. In simple terms, it allows you to look at protein-protein interactions with the help of fluorescently labeled proteins.
In not so simple terms…
…it allows you to quantitatively measure protein interactions using the process of energy transfer from a fluorophore that’s in an excited state (donor) to a neighboring fluorophore (acceptor) through dipole-dipole interactions. For this transfer to happen:
- the distance between the two proteins must be less than 10nm or 100 angstroms (Å)
- there must be overlap between the emission dipole of the donor and the absorption dipole vector of the acceptor
- and the emission spectrum of the donor must overlap with the excitation spectrum of the acceptor
If FRET occurs successfully, then the intensity of the donor fluorescence will decrease and the acceptor fluorescence will increase. This is called donor quenching, and shows you that there has been some kind of interaction between the two proteins.
Considering that protein interactions play a key role in most signaling pathways, combined with the fact that this can be done with live cells, it is certainly an appealing tool. For a more detailed look at the ins and outs of FRET including all the equations you may need, I recommend looking at Peter Nagy’s tutorial.
FRET + FACS = FCET
Flow cytometric FRET, also known as FCET, can measure both the decrease of fluorescence in the donor (the donor quenching mentioned above) and the increase in fluorescence in the acceptor (called sensitized emission). The main advantage of combining FRET with FACS is that you can look at large populations of cells over a short time-period, but still retain resolution of data to the single-cell level.
Labeling your proteins
For this technique the proteins of interest can be labeled using a few different methods. The most straight-forward method of labeling is using fluorophore-conjugated antibodies, and if possible, direct labeling with conjugated monoclonal primary antibodies. Indirect labeling adds in unwanted layers that could lead to greater separation of the fluorophores and thus diminish FRET. You can also label your proteins of interest with fluorescent tags. If you choose these, they should be small in size, bright and stable, and not cause any change to the cell into which it is introduced.
The labeling you choose may limit the analysis you can do, for example using some antibodies may limit you to extracellular molecules. There are too many different types of label to go into here, but you can find some useful information in this guide, in addition to the ones noted below.
A few quick pointers for FCET:
- Suspended cells are easier to work with – this is a general rule in FACS too. If you use adherent cells, extra steps will have to be added; care should be taken not to damage the cells in this case.
- Choose your fluorophores wisely, whichever way you label your cells (antibodies or labeling) they have to be detectable by your machine and they have to overlap in a specific way (see above). Some examples of common pairs of FCET/FRET are:
- Alexa488/Cy2 and Alexa546/555
- CFP and YFP
- GFP and RFP
- Make sure you have had a tour of the evaluation program you need to use. There may be specific settings you need to know beforehand.
If you have checked out the tutorial I mentioned above, or even looked up some basic FRET papers, you will know that it could appear very complicated. There are many ways by which to quantify FRET, although many of these remain theories and are rarely used. Some also require the use of microscope-based FRET. For those of you that would like more detail there is a great chapter in Springer’s Flow Cytometry Protocols, which is part of Springer Protocols, Methods in Molecular Biology. It goes in to much more depth and offers methods too.
Finally, don’t be disheartened if it doesn’t work the first time. Like any experiment it can take a lot of optimization.
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