Game 6: Monocyte Background
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It has been shown(1) that some cyanine (Cy™)—and cyanine-like—dyes can nonspecifically bind to monocytes. Additional nonspecific binding to the human Fc receptor has been observed. Nonspecific monocyte binding can decrease data resolution by increasing background and/or causing a false positive signal.
In this Fluorochrome Faceoff, we evaluate dyes excited by the yellow-green laser with peak emission around 780 nm on several clones.
As expected, PE-Cy7 demonstrated significant non-specific monocyte binding, irrespective of the antibody clone it was conjugated to. Bio-Rad StarBright™ Yellow 800 and BioLegend PE/Fire™ 810 also demonstrated varying degrees of nonspecific binding to the monocyte population.
Human whole blood was stained with BD Horizon™ RY775 Mouse Anti-Human CD3 UCHT1 (Cat. No. 571376), BD Horizon™ RY775 Mouse Anti-Human CD8 RPA-T8 (Cat. No. 571380), BD Horizon™ RY775 Mouse Anti-Human CD19 SJ25C1 (Cat. No. 571374), BD Pharmingen™ PE-Cy7 Mouse Anti-Human CD3 UCHT1 (Cat. No. 563423), BD Pharmingen™ PE-Cy7 Mouse Anti-Human CD8 RPA-T8 (Cat. No. 557746), BD Pharmingen™ PE-Cy7 Mouse Anti-Human CD19 SJ25C1 (Cat. No. 557835), Bio-Rad StarBright™ Yellow 800 Mouse Anti-Human CD3 UCHT1 or BioLegend PE/Fire™ 810 Mouse Anti-Human CD19 SJ25C1 Reagent. Erythrocytes were lysed with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899). The bivariate pseudocolor plots showing the expression of CD3, CD8 and CD19 versus side light-scatter (SSC-A) signals were derived from gated events with the forward and side light-scatter characteristics of viable leukocytes. Flow cytometry and data analysis were performed using a BD FACSymphony™ A5 SE Cell Analyzer System and FlowJo™ Software. N/A means the corresponding clone/specificity was unavailable conjugated to that fluorochrome at the time of our testing.
Winner’s Circle
BD Horizon RealYellow™ 775 was the only fluorochrome that did not demonstrate nonspecific background binding to monocytes and is declared the winner of this Fluorochrome Faceoff!
PRO TIP: There are several different blocking buffers available to help improve these nonspecific interactions, such as the BD Pharmingen™ MonoBlock™ Leukocyte Staining Buffer (Cat. No. 570002).
Runner-Up:
SBY800
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.
BD flow cytometers are Class 1 Laser Products. For Research Use Only. Not for use in diagnostic or therapeutic procedures.
CF is a trademark of Biotium, Inc. Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.