Learn to Love Tandem Dyes
If you design staining panels with upwards of 5 or 6 fluorescent parameters, you likely experience the love-hate relationship that many researchers have with tandem dyes. These frequently maligned fluorochromes do require a little more care and consideration than the seemingly indestructible small molecule fluorochromes, but with a little effort, that love-hate relationship can tilt toward the love side! As described in our blog article on "Tandem Dye Myths Debunked", tandem dyes do not ‘fall apart’, but conditions such as light exposure and bubbly buffers may oxidize the dye which leads to a loss of FRET efficiency and, hence, more fluorescence from the donor dye.
Learn to Love Tandem Dyes Rule #1 - Avoid elevated temperatures and extended light exposure.
Elevated temperatures and extended exposure to light are probably the worst conditions to subject a tandem dye to but as these are unavoidable in normal work flow, it is important to minimize the time of exposure.
To illustrate, we pulled retained samples for PE-Cy7 conjugated anti-Human CD4 (clone RPA-T4) and used them to stain freshly isolated PBMC – the data are shown in part A of the figure below, including the compensation required to remove PE-Cy7 signal from the PE detector. These samples represented:
- Four different lots, 1 each year from 2007-2010
- 4 lots had been stored at 4°C since production.
- Each vial was opened only a handful of times since their initial production.
These data demonstrate that PE-Cy7 is stable if you always keep it at 4°C and only open the vial once or twice a year…..but that’s just silly!
So, one of the most important things you can do is educate your lab colleagues (or anyone else that might borrow your reagents) that they need to minimize the exposure of tandem dye reagents to light when removing the material needed for staining from the reagent vial. Remember, it’s all for one, and one for all in this matter! If just one of person leaves an open vial sitting on the bench, then everyone using that vial will suffer the consequences.
The consequences are illustrated in part B of the figure below where cells are stained with RPA-T4 PE-Cy7 and acquired either immediately (pink histogram) or after 6 hours of exposure to ambient light (blue histogram). The actual PE-Cy7 signal is not impacted by the light exposure (left) but you can see the increase in fluorescence from the tandem in the PE detector (right) due to the loss of FRET efficiency after extended exposure to light.
Learn to Love Tandem Dyes Rule #2 - Always use high quality formaldehyde for fixation.
Another common complaint associated with tandem dyes is they don’t "hold up well" using fixation conditions. The actual issue is a loss of FRET efficiency after fixation, aka increased fluorescence from the donor part of the tandem leading to higher compensation required.
High-quality formaldehyde with minimal metallic impurities helps minimize loss of FRET efficiency. We also recommend fixing your cells for 20-30 minutes in a 2% (final concentration) formaldehyde solution at room temperature in the dark, then wash away the formaldehyde. We encourage you not to leave cells sitting in formaldehyde any longer than overnight.