BD Horizon RealBlue™ 780 (RB780) Reagents leverage an innovative laser-specific fluorochrome, excited primarily by the 488-nm blue laser to offer:
- Minimal cross-laser excitation off the 561-nm yellow-green laser
- A bright fluorochrome to support the detection of low-expression surface and intracellular markers
- Reduced monocyte background compared to PE-Cy7
RB780 Reagents Can Be Used with PE-Cy7 on Appropriately Configured Conventional and Spectral Flow Cytometers
| Format | Ex Max | Em Max | Spectral | Conventional | Relative Brightness | Spillover* (1 = low, 4 = high) | Alternative To |
|---|---|---|---|---|---|---|---|
| RB780 | 488 nm | 780 nm | ✓ | ✓ | 1 | PE-Cy7, PerCP/Fire™ 780, BB790-P |
*Value may vary based on instrument configuration and settings. Spillover ranking is based on cross-laser excitation on five-laser spectral instruments and does not take into account spillover into adjacent detectors.
Performance
BD Horizon RealBlue™ 780 Reagents Provide Minimal Cross-laser Excitation Off the 561-nm Yellow-Green Laser and Reduced Background Compared to PE-Cy7
RB780 has minimal cross-laser excitation
Normalized emission profiles of RB780 and PE-Cy7 fluorochromes. Human CD4 SK3 antibody acquired on a BD FACSDiscover™ S8 Cell Sorter.
Compared to PE-Cy7, RB780 shows lower spillover spread into detectors on the yellow-green laser
Human whole blood was stained with BD Horizon™ RB780 Human CD4 (SK3) Reagent, acquired and compensated on a BD FACSymphony™ A5 SE Cell Analyzer. The biexponential scaling was set for PE-Cy7 and applied to RB780 to highlight changes in spread into selected channels.
BD Horizon™ RB780 Reagents offer reduced background compared to PE-Cy7
Human whole blood was stained with PE-Cy7 or BD Horizon™ RB780 conjugated to human CD3 (UCHT1) Reagent, CD4 (SK3), human CD8 (RPA-T8), or human CD19 (SJ25C1), followed by lysis with BD FACS™ Lysing Solution. All specificities are analyzed with the B810 detector on a BD FACSymphony™ A5 SE Cell Analyzer.
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Applications
Staining of PE, PE-Cy7 and BD Horizon™ RB780 Mouse Anti-Human CD279 (PD-1) Reagents. PE is detected in the YG585 detector, and both PE-Cy7 and RB780 are detected with the B810 detector. Data were acquired and compensated on a BD FACSymphony™ A5 SE Cell Analyzer.
Human PBMCs were stimulated with PMA and ionomycin in the presence of BD GolgiStop™ Protein Transport Inhibitor for 5 hours (top) or left unstimulated (bottom) and then stained with either BD Horizon™ RB780 (left, 0.125 µg) or PE-Cy7 (right, 0.25 µg) IFN- γ (clone B27) and matched isotype controls. Samples were acquired on a BD FACSymphony™ A5 SE Cell Analyzer, detected with the B810 detector.
Bivariate overlay plots of spectrally unmixed single color CD4 (with either RB780 or PE-Cy7) stained PBMCs obtained from healthy donor. Samples were acquired on the BD FACSymphony™ A5 SE Cell Analyzer and spectrally unmixed using FlowJo™ v10.8 Software.
BD Horizon™ RB780 Reagents show stable performance with lot-to-lot consistency across made-to-stock reagents and BD OptiBuild™ On-Demand Reagents. Human whole blood was stained with human CD4 (SK3) RB780 using several different dye lots (left), followed by lysis with BD FACS™ Lysing Solution. Mouse splenocytes were stained with mouse CD8a 53-6.7 using six different dye lots (right). All specificities were run on a BD FACSymphony™ A5 SE Cell Analyzer.
Multicolor Flow Cytometry
BD Horizon™ RB780 Reagents Resolve Well in a 12-color T Cell Inhibitory Panel Acquired on the BD FACSLyric™ Flow Cytometry System
Gating scheme of T cell subsets
Healthy PBMC were stained with a 12-color inhibitory T cell panel and acquired on the BD FACSLyric™ Flow Cytometry System. Labels on top of each plot show the parental gate from which plots are derived; numbers in each quadrant represent the frequency of cell population; T cell subsets are identified as N, naïve; CM, central memory; EM, effector memory; EMRA, effector memory RA+; and SCM, stem cell like memory.
RB780 Reagents and PE-Cy7 Can Be Used Together in Spectral Flow Cytometry
RB780 reagents and PE-Cy7 are used together in a multicolor panel to identify rare cell populations
BD Horizon™ RB780 Reagents and PE-Cy7 can be used together in a multicolor panel to identify rare cell populations. CD123 BD Horizon™ RB780 Reagent and CD303 PE-Cy7 co-expresses on a sub-population of human plasmacytoid dendritic cells (pDC). Flow plots show clear distinction of the pDC population marked as CD123+ CD303+ cells derived from lineage negative CD14-CD16-, HLA-DR+ CD11c- cell populations in healthy PBMC samples. Samples were stained with the panel reagents along with Human BD Fc Block™ Reagent and analyzed on a BD FACSymphony™ A5 SE Flow Cytometer. FlowJo™ v10.8 Software was used for spectral unmixing and analysis.
Buffer Compatibility
BD Horizon RealBlue™ RB780 Reagents Are Compatible with a Broad Range of Fixation and Permeabilization Systems
| Buffers | Results |
| BD FACS™ Lysing Solution and BD Pharm Lyse™ Lysing Buffer | Compatible |
| CellBlox™ Blocking Buffer | Compatible |
| BD Cytofix™ Fixation Buffer | Stable at least 24 hours |
| 1% PFA | Stable at least 24 hours |
| BD Cytofix/Cytoperm™ Fixation and Permeabilization Solution | Compatible with antibody staining before and after fixation |
| BD FACS™ Permeabilizing Solution 2 | Compatible with antibody staining before and after fixation |
| BD Phosflow™ Perm Buffer III | Compatible with antibody staining before and after fixation |
| EDTA and Heparin | Compatible |
| BD Horizon™ Brilliant Stain Buffer (BSB) | Compatible |
BD flow cytometers are Class 1 Laser Products. For Research Use Only. Not for use in diagnostic or therapeutic procedures.
The BD FACSLyric™ Flow Cytometer is for Research Use Only with BD FACSuite™ Application for up to 12 colors. 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. CellBlox is a trademark of Thermo Fisher Scientific.