PE is excitable by the 488 nm laser, 532 nm and 561 nm lasers, therefore PE and its tandem dyes appear here and on the 488 nm excitable fluorophores web page. As PE is maximally excited at 546 nm (compared to 496 nm using the 488 nm laser), where possible it is advisable to use the 532 nm or 561 nm laser to get the maximal signal. This also means there is no compensation to perform between PE and FITC, two of the most commonly used fluorophores, which frees up space on the 488 nm laser to add more fluorophores.
Table 1. 532 nm and 561 nm excitable dyes for flow cytometry.
Fluorophore |
Ex max |
Em max |
Brightness |
ZE5 Filter |
Similar |
---|---|---|---|---|---|
PE |
546 |
578 |
5 |
577/15 |
SBY575, DyLight550 |
StarBright Yellow 575 |
548 |
579 |
3 |
577/15 |
PE, DyLight550 |
StarBright Yellow 605 |
572 |
606 |
3 |
615/24 |
PE-Dazzle594, PE-DyL594 |
PE-A647 |
546 |
667 |
4 |
670/30 |
PE-Cy5, SBY665 |
StarBright Yellow 665 |
554 |
670 |
3 |
670/30 |
PE-Cy5, PE-A647 |
PE-Cy5 |
546 |
667 |
5 |
670/30 |
PE-A647,SBY665 |
PE-Cy5.5 |
546 |
695 |
4 |
720/60 |
SBY720 |
StarBright Yellow 720 |
549 |
719 |
4 |
720/60 |
PE-Cy5.5, PE-Fire700 |
PE-A750 |
546 |
779 |
4 |
750LP |
PE-Cy7, SBY800 |
PE-Cy7 |
546 |
785 |
4 |
750LP |
PE-A750,SBY800 |
StarBright Yellow 800 |
549 |
788 |
4 |
750LP |
PE-Cy7, PE-A750 |
Phycoerythrin is a member of the light harvesting phycobiliproteins found in red algae. PE is one of the brightest fluorophores, but is rapidly photobleached, as are tandems of PE, making them unsuitable for fluorescent microscopy applications.
Fig. 1. PE staining. Human peripheral blood was stained with CD4 (MCA1267PE) and CD3 (MCA463A700) after incubation with 10% human serum to detect helper T cells. Data was acquired on the ZE5 Cell Analyzer.
StarBright Yellow 575 Dye is part of the StarBright Dye range, a new range of fluorescent nanoparticles specifically designed for flow cytometry to be bright with narrow excitation and emission profiles. Excitable exclusively by the yellow, 561 nm laser and emitting at 579 nm, this bright fluorophore is the ideal dye to replace PE in panels where the 488 nm laser is being used.
StarBright Yellow 605 Dye is part of the StarBright Dye range. Excitable exclusively by the yellow, 561 nm laser and emitting at 606 nm, this bright fluorophore is the ideal dye to replace PE-DyL594 and PE-Dazzle in panels where the 488 nm laser is being used.
PE-A647 is a tandem dye of PE and Alexa -Fluor 647, exclusive to Bio-Rad. It cannot be used with PE-Cy5 as it has similar excitation and emission characteristics. However, it is a good alternative as it does not exhibit the nonspecific binding to Fc receptors, particularly on monocytes, seen with PE-Cy5.
Fig. 2. PE-Alexa Fluor 647 staining. Murine bone marrow was stained with CD11b (MCA711P647) and GR-1 (MCA2387A488) after incubation with 10% mouse serum to identify monocytes and granulocytes. Data acquired on the ZE5 Cell Analyzer.
StarBright Yellow 665 Dye is part of the StarBright Dye range. Excitable exclusively by the yellow, 561 nm laser and emitting at 670 nm, this bright fluorophore is the ideal dye to replace PE-Cy5 in panels. As it is not a traditional tandem it does not have excitation by the 488 and 640 lasers of the donor and acceptor dyes.
PE-Cy5 is a tandem of PE and Cyanine 5. It cannot be used with PE-A647 or APC due to similar emission spectra. Cy5 in this tandem has been shown to bind nonspecifically to Fc receptors.
PE-Cy5.5 is a tandem of PE and Cyanine 5.5 which has been shown to have less nonspecific Fc receptor binding.
StarBright Yellow 720 Dye is part of the StarBright Dye range. Excitable exclusively by the yellow, 561 nm laser and emitting at 719 nm, this bright fluorophore is the ideal dye to replace PE-Cy5.5 in panels. It has reduced 488 nm laser excitation and unlike Cy-based dyes does not bind to monocytes.
PE-A750 is a tandem dye of PE and Alexa-Fluor 750, exclusive to Bio-Rad. It cannot be used with PE-Cy7 as it has similar excitation and emission characteristics. However, it is a good alternative as it is more stable and not as sensitive to paraformaldehyde fixation as PE-Cy7.
PE-Cy7 is a tandem of PE and cyanine 7. PE-Cy7 is very photosensitive and can degrade with paraformaldehyde fixation. It cannot be used with PE-A750 due to similar emission spectra.
StarBright Yellow 800 Dye is part of the StarBright Dye range. Excitable exclusively by the yellow, 561 nm laser and emitting at 788 nm, this bright fluorophore is the ideal dye to replace PE-Cy7 in panels. It has reduced excitation by the 488 laser and does not bind non-specifically to monocytes, unlike PE-Cy7.
The 532 nm and 561 nm lasers are just two of the common lasers found on flow cytometers. Bio-Rad has dyes excitable by most of the common lasers used in flow cytometry, details of which can be found on our Fluorophores for Flow Cytometry page.
For more information about fluorophores and immunophenotyping, refer to our flow cytometry resources below.