Game 1: Resolution
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Fluorochrome resolution is the degree of separation between the negative and positive populations as calculated by Stain Index,
(MFIpos − MFIneg)/2(rSDneg). CD4 is commonly used as a reference for Stain Index because of its robust expression and regular availability of the same clone in different formats. Here we measure CD4 (SK3) Stain Index values for several fluorochromes excited by the blue-laser with a peak emission around 780 nm.
Human whole blood was stained with BD Horizon™ RB780 Mouse Anti-Human CD4 SK3 (Cat. No. 568675), BD Pharmingen™ PE-Cy7 Mouse Anti-Human CD4 SK3 (Cat. No. 557852), BioLegend PerCP/Fire™ 780 Mouse Anti-Human CD4 SK3 or Cytek cFluor® BYG781 Mouse Anti-Human CD4 SK3 Reagent. Erythrocytes were lysed with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899). The bivariate pseudocolor plots showing the expression of CD4 versus side light-scatter (SSC-A) signals were derived from gated events with the forward and side light-scatter characteristics of viable leukocytes. Histograms were derived from gated events based on light scattering characteristics for lymphocytes. Flow cytometry was performed conventionally using a BD FACSymphony™ A5 SE Cell Analyzer System. Data were generated from the Blue 810 detector using FlowJo™ Software.
Winner’s Circle
While all the fluorochromes used in this experiment provide good population resolution of the CD4 positive cells, there are some differences in terms of the stain index values. We, therefore, ranked RB780, cFBYG781 and PE-Cy7 as the winners, with PerCP/Fire™ 780 as the runner up.
PRO TIP: Be sure to pair bright dyes (high CD4 stain index values) with lower antigen-expression markers to get the best possible biological resolution of your data!
Runner-Up:
PerCP/Fire™ 780
Try the Winning Fluorochrome
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Disclaimer: Results and conclusions shown throughout the Fluorochrome Faceoff are based on experiments performed under the conditions described. Users should evaluate reagents with their specific protocols as results may vary with different experimental conditions.
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