How can I set up compensation for tandem dyes in flow cytometry?

Tandem dyes work based on the mechanism of fluorescence-resonance energy transfer (FRET), whose efficiency (a measure of the transfer of energy from the donor to the acceptor) is never 100%. Hence, there’s always some level of spillover in the donor (parent ?uorochrome) channel, which makes compensation extremely important and necessary.

General compensation protocols could be followed to compensate tandem dyes, i.e. running single stained compensation controls and then work out compensation with the aid of software available on hand. However, cautions need to be taken when handling tandem reagents and preparing compensation controls.

1)Tandem dyes are relatively less stable compared to their corresponding individual dye components. A high level of spillover, which may exceed 100%, can be observed during compensation if the dyes are degraded. To avoid dye degradation, samples should be analyzed as soon as possible, and exposure to light and formaldehyde-based fixatives should be minimized.

2) The energy transfer efficiency from donor to acceptor in tandem dyes tends to be different each time the conjugation chemistry is performed. Therefore, the tandem dye conjugated antibody used in the experiment should be the same as the one used for compensation. Using a different lot of tandem dye or antibody could lead to inaccuracies in compensation calculations.