Fluorophores for the Yellow (561 nm) Laser

Overview

PE is excitable by both the 488 nm laser and 561 nm laser, 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 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. 561 nm excitable dyes for flow cytometry.

Fluorophore

Ex max

Em max

Brightness

ZE5 Filter

Similar

PE

546

578

5

577/15

DyLight550

PE-A647

546

667

4

670/30

PE-Cy5

PE-Cy5

546

667

5

670/30

PE-A647

PE-Cy5.5

546

695

4

720/60

 

PE-A750

546

779

4

750LP

PE-Cy7

PE-Cy7

546

785

4

750LP

PE-A750


PE (ex 546/em 578)

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.
 


 
PE (ex 546/em 578)
Fig. 1. PE staining.

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.


PE-A647 (ex 546/em 667)

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.
 


 
PE-A647 (ex 546/em 667)
Fig. 2. PE-Alexa Fluor 647 staining.

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.


PE-Cy5 (ex 546/em 667)

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 (ex 546/em 667)

PE-Cy5.5 (ex 496/em 695)

PE-Cy5.5 is a tandem of PE and Cyanine 5.5 which has been shown to have less nonspecific Fc receptor binding.
 


 
PE-Cy5.5 (ex 496/em 695)

PE-A750 (ex 496/em 779)

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 (ex 496/em 785)

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.
 


 
PE-Cy7 (ex 496/em 785)

​Fluorophores by Laser

The 561 nm laser is one of many common lasers found on flow cytometers. Bio-Rad has dyes excitable by 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.