The 488 nm laser go-to dye: PerCP tandems
Virtually all flow cytometers are equipped with a blue (488 nm) laser and configured to measure fluorescence in the conventionally known ‘FL3’ detector. Over the years, a number of fluorochromes have been introduced for use with this detector including the PE tandem dyes, PE-Cy5 and PE-Cy5.5, as well as the small fluorescent protein Peridinin chlorophyll (PerCP) and its tandem dyes, PerCP-Cy5.5 and PerCP-eFluor® 710. So many choices!
Peak Emission Wavelength
Initially, PE-Cy5 was the reagent of choice for the FL3 detector on instruments equipped with a single laser. However, the introduction of instruments equipped with a second red (633 nm) laser reveals problems with this fluorochrome as the Cy5 portion of the tandem itself is excited by the red laser. Because the emission wavelength of Cy5 is close to the emission of APC, compensation can often be problematic should both fluorochromes be used together.
Subsequently, the PE-Cy5.5 tandem and PerCP fluorochromes found favor with users of multi-laser instruments. The PE-Cy5.5 tandem continues to have fluorescence spillover into the PE detector while PerCP is somewhat dim in comparative fluorescence and subject to photobleaching when exposed to high-powered lasers. These problems are overcome with the development of PerCP tandem dyes which eliminate the spectral overlap with PE and are more photostable as a result of the tandem dye structure.
For the blue, 488 nm laser, we recommend the PerCP tandem dyes (PerCP-Cy5.5 and PerCP-eFluor 710) for the FL3 detector:
- Compensation in the PE detector is not required as PE is not the donor molecule in the tandem.
- Signal to noise resolution is as good as or better than PE tandem dyes.
- No cross-beam compensation required if the red laser is used at the same time.
- Stable fluorescence after fixation of cells.
Why PerCP-eFluor 710 is the best fluorochrome choice for FL3
- PerCP-eFluor 710 is two- to three-fold brighter than PerCP-Cy5.5 as shown below for anti-mouse CD4 (clone RM4-5) and anti-human CD3 (clone OKT3).
- PerCP-eFluor 710 easily resolves dim targets such as transcription factors as shown here for Eomes expression in mouse NK and CD8 cells.
- Currently, eBioscience offers almost 80 PerCP-Cy5.5 conjugates and nearly 200 PerCP-eFluor 710 conjugates. We continue to develop PerCP-eFluor 710 conjugated antibodies as it is a favorite choice of our customers.
Compare for yourself!
The following is a list of clones that eBioscience offers in both PerCP-Cy5.5 and PerCP-eFluor 710 formats for individual comparison: