FRET efficiency is never 100%, which means that there will always be some bleedthrough emission from the donor that is observed. This is dependent on the quality of the tandem conjugates. Be sure to use isotype controls and single color controls to appropriately adjust your compensation settings.
Tandems are commonly known for ”degradation“ or ”decoupling“, describing the loss of emission from the acceptor and increased emission by the donor. Because tandems are covalently attached, the donor and acceptor do not typically come apart in solution. There are potentially several causes that induce the loss of FRET to the acceptor. The first is exposure to light which is known to photobleach both the donor/acceptor molecules. Oxygen radicals also cause damage to the acceptor dyes. Both conditions would be irreversible and would confer loss of function to the tandem.
When using tandems in multicolor flow cytometry, it is critical to use isotype controls for background, single color controls for compensation, and FMO (Fluorescence-minus-one) controls for gating.
Secrets to successful tandem use:
Avoid extended exposure to light and extreme variations in temperature. Light will photobleach your tandem – protect your samples and antibody from light at all times. Do not freeze your tandem antibody conjugates, as this could potentially lead to denaturing of the donor protein.
Avoid ”overfixing“. We recommend that tandems be fixed for short-periods of time (less than 30 minutes) and then washed and resuspended in Cell Staining buffer for storage. For this purpose, we provide specially formulated FluoroFix™ buffer, for the fixation of tandem dyes.
Be aware of cross-beam excitation of your acceptor molecule when performing multi-laser flow cytometry with tandems. In most cases, this can be compensated. For example, the Cy5 acceptor in PE/Cy5 is known to be excitable by the red laser, so use caution in combination with APC.
Label your cells at 4°C or on ice. APC tandems may be susceptible to cell-mediated decoupling, so slowing down cell metabolism at low temperatures can help preserve the stability of the tandem.