red-laser-230x195

Flow cytometry analysis with a red laser

Red light-excitable fluorophores play a critical role in standard multiparametric flow cytometry. With the growing availability of flow cytometers equipped with UV, violet, green, yellow, blue, and red lasers, the red laser continues to be an essential part of many flow cytometry instruments; additionally, red laser-excitable fluorophores are commonly used because of compatibility with Green Fluorescent Protein for live-cell analysis. The expansive Alexa Fluor dye, eFluor dye, and NovaFluor dye portfolios support multi-color immunophenotyping by flow cytometry and include a variety of dyes compatible with the 633 nm spectral line of the red laser.

Properties of fluorescent dyes for the red laser

The table below lists the properties of the most commonly used fluorophores for the red laser.

FluorophoreFluorophore typeEx/Em (nm)Relative intensity*
(1 = lowest, 5 = highest)
Suggested bandpass filter (nm)Uses
Allophycocyanin (APC)Protein645/6605660/20Widely used fluorophore, APC is an intensely bright phycobiliprotein isolated from red algae that exhibits far-red fluorescence with high quantum yields; alternatives are Alexa Fluor 647, Cyanine 5, and eFluor 660 dye
NovaFluor Red 660Phiton technology637/6593660/20Alternative to APC and Alexa Fluor 647 dye, with a more narrow emission spectrum
Alexa Fluor 647Organic dye650/6685660/20Widely used alternative to APC; photostable, making it a great choice for microscopy, in addition to flow cytometry
eFluor 660Organic dye633/6685660/20Photostable, making it a great choice for microscopy, in addition to flow cytometry; alternative to APC or Alexa Fluor 647 dye
Cyanine 5Organic dye649/6663660/20Good choice for intracellular labeling and microscopy; alternative to APC and Alexa Fluor 647 dye
NovaFluor Red 685Phiton technology637/6854695/40Relatively brighter dye useful in complex multi-color flow cytometry panels; NovaFluor Red 685 dye can be used to fill the spectral gap between Alexa Fluor 647 and Alexa Fluor 700 dyes
Alexa Fluor 660Organic dye662/6904695/40A unique signature optimized for flow cytometers with spectral capabilities, to fit between fluorophores Alexa Fluor 647 and Alexa Fluor 700 in complex multi-color panels
APC-Cyanine 5.5Tandem645/6943695/40Tandem dye, resonance energy transfer from APC molecule to Cyanine 5.5 dye
NovaFluor Red 700Phiton technology639/7004695/40A brighter dye useful for complex multi-color flow cytometry panels, NovaFluor Red 700 dye can be used to fill the spectral gap between Alexa Fluor 647 and Alexa Fluor 700 dyes
NovaFluor Red 710Phiton technology639/7104695/40Can be used as an alternative to Alexa Fluor 700 and APC-Alexa Fluor 700, with less cross-laser excitation and spectral spillover
Alexa Fluor 700Organic dye696/7192720/30Although not optimally excited by the red laser, it produces a bright signal that can be used as an additional dye with this laser
NovaFluor Red 725Phiton technology636/7254720/30A very low spillover, bright, and spectrally unique dye that peaks in the R5 detector of the Cytek Aurora. Ideal for filling in gaps in spectral panels.
NovaFluor Red 755Phiton technology636/7254780/60The only red laser–excited commercial dye that has peak emission in the R6 detector of the Cytek Aurora. A stable and bright non-tandem alternative to APC-Cyanine 7.
APC-Cyanine 7Tandem645/7674780/60Tandem dye, resonance energy transfer from APC molecule to Cyanine 7 dye; APC-Cyanine 7, APC-Alexa Fluor 750, and APC-eFluor 780 may be used interchangeably
APC-eFluor 780Tandem645/7672780/60Tandem dye, resonance energy transfer from APC molecule to eFluor 780; more photostable than APC-Cyanine 7; APC-Cy 7, APC-Alexa Fluor 750, and APC-eFluor 780 may be used interchangeably
APC-Alexa Fluor 750Tandem645/7754780/60Tandem dye, resonance energy transfer from APC molecule to Alexa Fluor 750; APC-Cy 7, APC-Alexa Fluor 750, and APC-eFluor 780 may be used interchangeably
* The relative intensity of any fluorophore is dependent upon cell type being studied, reagent used, instrument, and instrument configuration. The relative intensities provided are to be used as a guideline; observed intensities may vary.

Support

Custom Services
Contact our eBioscience custom services group for antibody conjugation, antibody cocktails, immunoassay development, bulk reagents, and testing services.

For Research Use Only. Not for use in diagnostic procedures.