Tandem Dye Myths Debunked
Tandem dyes have been used in flow cytometry for decades and have allowed for progressively larger numbers of markers to be investigated in a single experiment. Myths about tandem dyes exist: we have all heard or said “the tandem fell apart” or there is lot-to-lot variability due to “inconsistency in the manufacturing process.” There are also some additional considerations for using tandem dyes in multi-color flow cytometric experiments. Before addressing these myths and considerations, a little background about tandems.
What is a tandem dye?
A tandem is composed of two fluorescent molecules (typically a large protein based dye-- the donor-- and a smaller organic dye-- the acceptor) covalently attached to each other that, through the phenomenon of Förster resonance energy transfer (FRET) also known as fluorescence resonance energy transfer, predominantly emit light characteristic of the more red emitting dye.
FRET efficiency is dependent upon:
- The overlap of the donor emission spectrum and the acceptor absorbance spectrum.
- The distance between the donor and acceptor (this typically needs to be <10 nm).
- The relative orientation of the donor and acceptor.
If the FRET efficiency is very high you will see a strong signal in your acceptor channel and weaker signal or bleed in your donor channel. If FRET efficiency is low you will see a strong signal in the donor channel and a weak signal in the acceptor channel. Since FRET efficiency will never reach 100% there will always be some bleed back into the channel of the donor dye.
Myth 1: Tandem dyes fall apart
How do tandem dyes fall apart? They don’t, because tandems are covalenty conjugated. When you hear someone say the “tandem fell apart” what they mean to say is there is very little or no signal in the acceptor dye channel and a very strong signal in the donor dye channel. This is usually due to a decrease in the FRET efficiency that can be caused by:
- Photobleaching of either the donor or acceptor dye
- This can occur if the tandem dye is not stored properly. These dyes are light sensitive. If prolonged exposure occurs it can result in decreased performance.
- Stability of the donor dye
- The donor dyes are all protein based and with time and/or improper storage conditions slight conformational changes can occur. Since FRET efficiency is related to both distance and orientation of donor and acceptor small changes (even a few nanometers) in secondary structure can significantly decrease FRET efficiency.
At eBioscience all tandems are put through a stringent development, validation, and stability process to ensure superior performance. If they are handled and stored as recommended there will not be any significant decrease in performance over the shelf life of the product (up to 2 years depending on format).
Myth 2: Lot-to-lot variability due to inconsistency in the manufacturing process
It is true that tandem dye conjugates are more complex to make than single dye conjugates. However, during development and validation, the manufacturing process is optimized: reproducibility standards are a requirement prior to commercialization. All lots must meet strict in-process specifications (protein dye/organic dye, FRET efficiency and F/P) in addition to flow performance specifications before they are released. If a lot or product does not meet these specifications, it is not released. This ensures lot-to-lot consistency.
PerCP-Cy5.5 Tandem Dye - CD3, clone SK7. Single donor stained with 3 different lots.
Considerations for using tandem dyes
When designing a flow cytometric experiment that incorporates tandem dyes it is important to remember the following:
- Compensation for the tandem dye should be set using the same antibody-conjugate as will be used in the experiment.
- If the target is dim or low in frequency, then compensation beads should be used.
- Samples should be protected from light at all times.
- Fixation and permeabilization, if required, should be done using high-quality reagents.
- Impurities may adversely affect FRET efficiency.
- Also avoid vortexing fixation and permeabilization buffers before use.
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