What is tandem dye breakdown? What measures should be taken to account for it?

A tandem dye is composed of two attached fluorescent molecules or fluorophores: one serves as the donor and the other as the acceptor. The donor fluorophore is excited by a light source and transfers its absorbed energy to the acceptor fluorophore, which then emits fluorescence. This transmission of energy is known as fluorescence resonance energy transfer (FRET).

A tandem dye may break down (or decouple/degrade) with extended exposure to light, drastic changes in temperature, or fixation. Light will photobleach both the donor and acceptor and this process is irreversible. Tandem dyes and stained samples should be protected from light at all times. Drastic changes in temperature may denature the donor fluorochrome and cause dimmer tandem dye expression. The brightness of tandem dyes may also be reduced by a fixation or permeabilization step. Fixed samples should be acquired as soon as possible.

Tandem dye breakdown results in having false positive signals in donor channels (see examples below) and might be mistaken for undercompensation. However, increasing the compensation between the channels does not remove the false positive signals, and may result in false negatives due to overcompensation.



It is important to recognize and be familiar with the breakdown patterns so that no false positives are made and defected antibody reagents are removed from use promptly.

Not all tandem dyes have the same degradation rates. PE-Cy7 conjugates are known to be very sensitive to light exposure, whereas PE-Cy5 and PerCP Cy5.5 conjugates are much more stable.

References:
1. Carter, A. Tandem Dye Breakdown. ICCS e-Newletter Vol. V No. 2, Spring 2014.
2. Hulspas A, Dombkowski D, Preffer F, Douglas D, Kildew-Shah B, Gilbert J. Flow cytometry and the stability of phycoerythrin-tandem dye conjugates. Cytotmetry Part A 2009;75A:966-972.