BD Horizon RealYellow™ 586 (RY586) Reagents leverage an innovative laser-specific fluorochrome, excited primarily by the 561-nm yellow-green laser to offer:
- Minimal cross-laser excitation off the 488-nm blue laser
- A bright fluorochrome to support the detection of low-expression surface and intracellular markers
- Significantly less cross-laser excitation compared to PE
- An alternative to PE on conventional flow cytometers
- Compatibility with PE on spectral flow cytometers equipped with both the 561-nm yellow-green and 488-nm blue lasers
RY586 Reagents Offer High Resolution with Minimal Cross-Laser Excitation
| Format | Ex Max | Em Max | Spectral | Conventional | Relative Brightness | Spillover* (1 = low, 4 = high) | Alternative To |
|---|---|---|---|---|---|---|---|
| RY586 | 565 nm | 586 nm | ✓ | ✓ | 1 | PE |
*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 RealYellow™ 586 Reagents Provide Minimal Cross-Laser Excitation Off the 488-nm Blue Laser Compared to PE
Normalized emission profiles of RY586 and PE fluorochromes. Human CD4 SK3 antibody acquired on a BD FACSymphony™ A5 SE Cell Analyzer.
RY586 offers lower spillover spread into select violet and UV channels compared to PE
Human whole blood was stained with BD Horizon™ RY586 Human CD4 (SK3) Reagent, acquired and compensated on a BD FACSymphony™ A5 SE Cell Analyzer.
Compared to PE, RY586 shows lower spillover spread into multiple detectors on the blue laser
Human whole blood was stained with BD Horizon™ RY586 Human CD4 (SK3) Reagent, acquired and compensated on a BD FACSymphony™ A5 SE Cell Analyzer. The biexponential scaling was set for PE and applied to RY586 to highlight changes in spread into selected channels.
Applications
Human whole blood was stained with RY586 (left) or PE (right, acquired off the yellow-green laser) CD25 (2A3) and acquired 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 and then stained with APC CD4 and either RY586 (left, 0.125 µg) or PE (right, 0.25 µg, acquired off the yellow-green laser) IL-17α (N49-653) and acquired on a BD LSRFortessa™ Cell Analyzer.
Molt-4 cells were permeabilized with ice-cold 70% ethanol, stained with RY586 (left) or PE (right, acquired off the yellow-green laser) Ki-67 (B56) and DAPI for DNA content, and then acquired on a BD LSRFortessa™ X-20 Cell Analyzer.
Human PBMCs co-stained with PE and BD Horizon™ RY586 Reagents. Data acquired and spectrally unmixed on a BD FACSymphony™ A5 SE Cell Analyzer.
BD Horizon™ RY586 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) RY586 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 several different dye lots (right). All specificities were run on a BD FACSymphony™ A5 SE Cell Analyzer.
Multicolor Flow Cytometry
BD Horizon™ RY586 Reagents Can be Used with PE on Spectral Flow Cytometers
RY586 and PE can resolve distinct populations in a multicolor spectral flow cytometry panel
RY586 provides comparable brightness to PE and is ideal for detecting low-expression markers. Using RY586 and PE in the same spectral flow cytometry panel enables high-dimensional analysis of more low-expression surface and intracellular markers. Here we demonstrate this with a multicolor panel using both RY586 and PE fluorochromes to characterize human immune cells expressing different levels of CD16. Cells were acquired on a BD FACSymphony™ A5 SE Cell Analyzer and analyzed with FlowJo™ Software. The spectral unmixing matrix contained 14 fluorochromes.
BD Horizon RealYellow™ 586 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 Horizon RealYellow™ 586 (RY586) Reagents look like the best alternative for PE and will simplify panel design on conventional flow cytometers. They can also be an additional dye for spectral panel design.”
Gert Van Isterdael, PhD
VIB Ghent University, Belgium
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.