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The eBioscience brand of fluorochromes. Learn more about
eFluor®.
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The eFluor® solution for the violet laser provides three fluorophores that work together to provide maximum resolution of antigenic determinants when used simultaneously.
The eFluor® solution includes one organic-based dye and two nanocrystal reagents. The superior performance of this reagent package is accomplished by narrow emission
peaks with little spectral overlap and is in stark contrast to the broad emission peaks and spectral overlap seen with the traditional approach for the violet laser.
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Emission spectra of traditional and eFluor® reagents for the violet laser |
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Normalized emission spectra of the traditional fluorophores commonly used with the violet laser (top) versus the eFluor® solution (bottom). The advantageous narrow emission
spectra of the eFluor® solution is evident when compared to the broad emission spectra and large spectral overlap of the traditional approach. The recommended band pass
filters for each fluorophore are indicated.
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To support violet laser-based multicolor flow cytometry, eBioscience has developed three eFluor® fluorophores compatible with the violet laser (405 nm) line. This reagent solution for
the violet laser includes eFluor® 450, an organic-based dye, and two nanocrystal-based reagents, eFluor® 605NC and eFluor®
650NC, that have been optimized for superior optical performance including high signal to noise ratios and minimal intra-laser compensation
requirements. The pairing of appropriate antibody specificities with each fluorophore will maximize the amount of information that can be acquired from a valuable sample.
eBioscience currently offers over 100 reagents optimized for use with the violet laser.
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eFluor® reagents for the violet laser
To illustrate the quality performance of eFluor® reagents for the violet laser, staining examples for each fluorophore are shown. Human peripheral blood cells were stained with
eFluor® 450 anti-human CD45RA or the same antibody clone conjugated to Pacific Blue® (left plot). Mouse splenocytes were stained with eFluor®
605NC anti-mouse CD8 or the same antibody clone conjugated to Pacific Orange® (middle plot) and eFluor®
650NC anti-mouse B220 or the same antibody clone conjugated to Qdot® 655; (right plot).
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The emission profiles of eFluor® 450, eFluor® 605NC, and eFluor® 650NC
are sufficiently narrow and well separated such that the intra-laser compensation required when using all three fluorophores simultaneously is less than 5% in any violet laser detector.
The resulting benefits of reduced compensation requirements include minimal data spread, resolution of dim and bright subpopulations and improved separation of positive and negative
populations as evidenced by calculating the relative stain index.
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A: Traditional Approach
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B: eFluor® Solution
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Comparison of the “traditional approach” with the eFluor® solution
Human PBMC were stained with the traditional fluorophores AmCyan anti-human CD3, Pacific Blue® anti-human CD4, and Pacific Orange® anti-human CD8 (A) or with the
eFluor® solution of eFluor® 650NC anti-human CD3, eFluor® 450 anti-human CD4, and eFluor®
605NC anti-human CD8 (B). The required compensation values for data plots are shown.
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Relative Stain Index
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Relative stain index of the eFluor® solution versus traditional fluorophores
From the data shown above, the relative stain index was calculated from the compensated data for each antibody used in the staining panels.
The percent improvement offered by the eFluor® reagents over the traditional fluorophores is shown for each specificity.
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Recommended configuration for detection of eFluor® fluorescence with a violet laser trigon
Picture of a trigon optical block (Becton Dickinson) showing the recommended configuration and filters for optimal detection of
eFluor® 450, eFluor® 605NC, and eFluor® 650NC following violet laser excitation.
Other systems may differ and will need to be configured according to manufacturers instructions.
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