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Primary Antibodies for the Blue Laser
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, eFluor dye, and NovaFluor dye portfolios support multi-color immunophenotyping by flow cytometry and include a variety of dyes compatible with the 488 nm spectral line of the blue laser.
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 |
|---|---|---|---|---|---|
| NovaFluor Blue 510 | Phiton technology | 496/511 | 2 | 506/18 | Dye with a unique spectra, compatible with other blue laser excitable dyes having a narrow emission spectrum. Ideal for spectral flow cytometry |
| 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 |
| NovaFluor Blue 530 | Phiton technology | 509/530 | 1 | 525/18 | A good alternative for other dyes that are excited by the 488 nm blue laser, such as FITC and Alexa Fluor 488 dye, with a similar excitation spectrum and more narrow emission spectrum; ideal for spectral flow cytometry |
| NovaFluor Blue 555 | Phiton technology | 496/555 | 1 | 542/18 | Excitation and emission profile similar to that of Alexa Fluor 532 dye, but brighter; ideal for spectral flow cytometry |
| NovaFluor Blue 585 | Phiton technology | 494/585 | 1 | 580/18 | Unique emission spectrum, which fits in between NovaFluor Blue 555 and NovaFluor Blue 610, allowing the detection of an additional marker in a complex multi-color panel; ideal for spectral flow cytometry |
| NovaFluor Blue 610-30S | Phiton technology | 509/614 | 1 | 610/20 | Can be used to replace some PE-tandem dyes with much less cross-excitation and lower spectral spillover, allowing detection of an additional marker labeled with NovaFluor Yellow 610 dye; NovaFluor Blue 610-30S is dimmer than NovaFluor 610-70S |
| NovaFluor Blue 610-70S | Phiton technology | 509/614 | 2 | 610/20 | Can be used to replace some PE-tandem dyes with much less cross-excitation and lower spectral spillover, allowing detection of an additional marker labeled with NovaFluor Yellow 610 dye; NovaFluor Blue 610-70S is brighter than NovaFluor Blue 610-30S |
| NovaFluor Blue 660-40S | Phiton technology | 509/614 | 1 | 660/20 | Can be used to replace PE-tandem dyes with much less cross-excitation and lower spectral spillover, allowing detection of an additional marker labeled with NovaFluor Yellow 660 dye; NovaFluor Blue 660-40S is dimmer than NovaFluor 660-120S |
| NovaFluor Blue 660-120S | Phiton technology | 509/614 | 3 | 660/20 | Can be used to replace PE-tandem dyes with much less cross-excitation and lower spectral spillover, allowing detection of an additional marker labeled with NovaFluor Yellow 660 dye; NovaFluor Blue 660-120S is brighter than NovaFluor 660-40S |
| 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 |
| NovaFluor Blue 690 | Phiton technology | 494/688 | 1 | 695/40 | Can be used to replace tandem dyes, such as PerCP-Cyanine5.5, with much less cross-excitation and lower spectral spillover. Ideal for spectral cytometry. |
| 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 |
| NovaFluor Blue 725 | Phiton technology | 492/725 | 3 | 710/50 | NovaFluor Blue 725 dye should generally be paired with moderately to highly expressed antigens in multicolor panels |
| NovaFluor Blue 760 | Phiton technology | 490/764 | 3 | 780/60 | NovaFluor Blue 760 dye should generally be paired with moderately to highly expressed antigens in multicolor panels |
| 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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