A Clean Dye for Spectral Flow Cytometry
BD Horizon RealBlue™ 545 (RB545) Reagents leverage an innovative laser-specific fluorochrome, excited primarily by the 488-nm blue laser that offer:
- Minimal cross-laser excitation off the 561-nm yellow-green laser
- A moderate brightness fluorochrome to support the detection of high-expression surface and intracellular markers
- Optimized compatibility with spectral flow cytometers
RB545 Reagents Are Optimized for Use in Spectral Flow Cytometry Panels
| Format | Ex Max | Em Max | Spectral | Conventional | Relative Brightness | Spillover* (1 = low, 4 = high) | Alternative To |
|---|---|---|---|---|---|---|---|
| RB545 | 496 nm | 545 nm | ✓ | _ | 1 | Spark Blue™ 550 |
*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™ RB545 Reagents Offer Minimal Cross-laser Excitation Off the Yellow-green Laser
RB545 reagents offer minimal cross-laser excitation
Normalized emission profile of the RB545 reagent with minimal excitation off the 561-nm yellow-green laser. Human CD4 SK3 antibody acquired on a BD FACSDiscover™ S8 Cell Sorter.
RB545 demonstrates low spillover spread into detectors on multiple lasers
Human whole blood was stained with BD Horizon™ RB545 Human CD4 (SK3) Reagent, acquired on a BD FACSymphony™ A5 SE Cell Analyzer and compensated using FlowJo™ Software.
Applications
Human PBMCs were stimulated with PMA and ionomycin in the presence of BD GolgiStop™ Protein Transport Inhibitor for five hours and then stained with either BD Horizon™ RB545 Reagent or FITC IFN-γ (B27) and matched isotype controls. Samples were acquired on a BD FACSymphony™ A5 SE Cell Analyzer.
Mouse splenocytes were stained with mCD4 (GK1.5) BD Horizon™ RB545 Reagent (left) or FITC (right) and acquired on a BD FACSymphony™ A5 SE Cell Analyzer.
Spectral unmixing of RB545 and FITC. Bivariate overlay plots of spectrally unmixed single color CD4 (with either RB545 or FITC) stained PBMCs obtained from a healthy donor. Samples were acquired on the BD FACSymphony™ A5 SE Cell Analyzer and spectrally unmixed using FlowJo™ v10.8 Software.
Human PBMCs were stained with two reagent pairs: moderately similar CD4 RB545/CD127 BB515 and highly similar CD4 FITC/CD127 BB515. Reduced resolution was observed with the highly similar pair, as shown in the bottom row (CD4 FITC/CD127 BB515). Samples were acquired using the BD FACSymphony™ A5 SE Cell Analyzer and analyzed with FlowJo™ Software.
RB545 reagents show stable performance with lot-to-lot consistency across made-to-stock reagents and BD OptiBuild™ On-Demand Reagents. They also offer proven photostability when exposed to typical lab lighting or dimmed core lab lighting. Human whole blood was stained with human CD4 (SK3) RB545 reagent, followed by lysis with BD FACS™ Lysing Solution using four different dye lots (left). Mouse splenocytes were stained with mouse CD8a (53-6.7) using four different dye lots (right). All specificities were run on a BD FACSymphony™ A5 SE Cell Analyzer.
Multicolor Flow Cytometry
RB545 and FITC Can Be Used Together on a Spectral Flow Cytometer
RB545 is distinguished from FITC in an 8-color TBNK panel acquired on a BD FACSymphony™ A5 SE Cell Analyzer
Human whole blood was stained with an 8-color TBNK panel and lysed with BD FACSLyse™ Lysing Buffer. Samples were then acquired on a BD FACSymphony™ A5 SE Cell Analyzer and spectrally unmixed using FlowJo™ Software. Reagents used included CD3 (UCHT1) BUV737, CD4 (SK3) RB545, CD8 (SK1) FITC, CD16 (3G8) APC, CD19 (SJ25C1) PE, CD20 (2H7) APC-H7, CD45 (HI30) BUV395 and CD56 (NCAM16.2) BV421.
BD Horizon RealBlue™ 545 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.
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.