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.
The table below lists the properties of the most commonly used fluorophores for the red laser.
|Fluorophore||Fluorophore type||Ex/Em (nm)||Relative intensity*|
(1 = lowest, 5 = highest)
|Suggested bandpass filter (nm)||Uses|
|Allophycocyanin (APC)||Protein||645/660||5||660/20||Widely 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 660||Phiton technology||637/659||3||660/20||Alternative to APC and Alexa Fluor 647 dye, with a more narrow emission spectrum|
|Alexa Fluor 647||Organic dye||650/668||5||660/20||Widely used alternative to APC; photostable, making it a great choice for microscopy, in addition to flow cytometry|
|eFluor 660||Organic dye||633/668||5||660/20||Photostable, making it a great choice for microscopy, in addition to flow cytometry; alternative to APC or Alexa Fluor 647 dye|
|Cyanine 5||Organic dye||649/666||3||660/20||Good choice for intracellular labeling and microscopy; alternative to APC and Alexa Fluor 647 dye|
|NovaFluor Red 685||Phiton technology||637/685||4||695/40||Relatively 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 660||Organic dye||662/690||4||695/40||A 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 5.5||Tandem||645/694||3||695/40||Tandem dye, resonance energy transfer from APC molecule to Cyanine 5.5 dye|
|NovaFluor Red 700||Phiton technology||639/700||4||695/40||A 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 710||Phiton technology||639/710||4||695/40||Can be used as an alternative to Alexa Fluor 700 and APC-Alexa Fluor 700, with less cross-laser excitation and spectral spillover|
|Alexa Fluor 700||Organic dye||696/719||2||720/30||Although 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 725||Phiton technology||636/725||4||720/30||A 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 755||Phiton technology||636/725||4||780/60||The 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 7||Tandem||645/767||4||780/60||Tandem 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 780||Tandem||645/767||2||780/60||Tandem 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 750||Tandem||645/775||4||780/60||Tandem 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.|
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