Flow cytometry analysis with a blue laser

Blue 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, red and blue lasers, the blue laser continues to be an essential part of nearly every flow cytometry instrument, and additionally blue laser-excitable fluorophores are commonly used because of compatibility with Red 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 488 nm spectral line of the blue laser.

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Properties of fluorescent dyes for the blue laser

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

Fluorophore Fluorophore type Ex/Em (nm) Relative intensity* (1 = lowest, 5 = highest) Suggested bandpass filter (nm) Uses
Alexa Fluor 488 Organic dye 490/519 3 530/30 A brighter alternative to FITC that has greater photostability, pH-independent fluorescence from pH 4 to 10, and almost the identical fluorescence spectra
Fluorescein (FITC) Organic dye 490/525 3 530/ 30 Widely used fluorophore that is prone to photobleaching and exhibits pH-sensitive fluorescence between pH 5–8
PerCP Protein 482/678 3 695/40 Peridinin chlorophyll protein complex (PerCP) is a protein with a large Stokes shift that is ideal for multiplex fluorescence detection
PerCP-Cyanine 5.5 Tandem 482/695 3 695/40 Tandem dye, resonance energy transfer from PerCP molecule to Cyanine 5.5 dye
PerCP-eFluor 710 Tandem 482/710 4 710/50 Tandem dye, resonance energy transfer from PerCP molecule to eFluor 710 dye; 2-3 fold brighter than PerCP-Cyanine 5.5 tandem
R-Phycoerythrin (R-PE, PE) Protein 496, 565/575 5 575/26 Widely used fluorophore with a high quantum yield that is ideal for detection of low-density antigens
PE-Texas Red Tandem 496, 565/615 3 590/40 Tandem dye, resonance energy transfer from PE molecule to Texas Red dye
PE-Alexa Fluor 610 Tandem 496, 565/628 3 590/40 Tandem dye, resonance energy transfer from PE molecule to Alexa Fluor 610 dye
PE-eFluor 610 Tandem 496,565/607 3 610/20 Tandem dye, resonance energy transfer from PE molecule to eFluor 610; alternative to PE-Texas Red dye
PE-Cyanine 5 (TRI-COLOR) Tandem 496, 565/667 3 670/14 Tandem dye, resonance energy transfer from PE molecule to Cyanine 5 dye
PE-Cyanine 5.5 Tandem 496, 565/694 4 695/40 Tandem dye, resonance energy transfer Cyanine 5.5 dye
PE-Alexa Fluor 700 Tandem 496, 565/723 3 695/40 Tandem dye, resonance energy transfer from PE molecule to Alexa Fluor 700 dye
PE-Cyanine 7 Tandem 496, 565/767 4 780/60 Tandem dye, resonance energy transfer from PE molecule to Cyanine 7 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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