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 and eFluor dye portfolios support multicolor immunophenotyping by flow cytometry and include a variety of dyes compatible with the 633 nm spectral line of the red laser.

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Step 1. Search for the marker of interest (e.g., “CD4” or “CD45”).
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Properties of fluorescent dyes for 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
APC Protein 645/660 5 660/20 Widely used fluorophore, allophycocyanin (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
APC-Alexa Fluor 750 Tandem 645/775 4 780/60 Tandem dye, resonance energy transfer from APC molecule to Alexa Fluor 750; APC-Cy7; APC-Alexa Fluor 750 and APC-eFluor 780 may be used interchangeably
APC-Cyanine 5.5 Tandem 645/694 3 695/40 Tandem dye, resonance energy transfer from APC molecule to Cyanine 5.5 dye
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-Cy7;
APC-Alexa Fluor 750 and APC-eFluor 780 may be used interchangeably
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
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 a third dye off this laser; please note that emission wavelength requires a distinct set of filters
Cyanine 5 Organic dye 649/666 3 660/20 Good choice for intracellular labeling and microscopy; alternative to APC and Alexa Fluor 647 dye
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
*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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