Superior Alternative to PE-Cy5
BD Horizon RealBlue™ 670 (RB670) Reagents leverage an innovative laser-specific fluorochrome, excited primarily by the 488-nm blue laser to offer:
- Reduced cross-laser excitation off the 561-nm yellow-green laser
- A bright fluorochrome to support the detection of low-expression surface and intracellular markers
- Improved spillover compared to PE-Cy5, NovaFluor™ Blue 660 and StarBright™ Blue 675
- Reduced monocyte background compared to PE-Cy5
RB670 Reagents Can Be Used in Place of or Along With PE-Cy5 on Appropriately Configured Cytometers
| Format | Ex Max | Em Max | Spectral | Conventional | Relative Brightness | Spillover* (1 = low, 4 = high) | Alternative To |
|---|---|---|---|---|---|---|---|
| RB670 | 492 nm | 670 nm | ✓ | ✓ | 2 | PE-Cy5, |
*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™ 670 Reagents Are a Cleaner Alternative to PE-Cy5, StarBright™ Blue 675 and NovaFluor™ Blue 660
RB670 reagents have reduced cross-laser excitation
Normalized emission profiles of CD4 (SK3) RB670, PE-Cy5 and NFB660-120S, and CD4 (RPA-T4) SBB675 fluorochromes. Data acquired on a BD FACSDiscover™ S8 Cell Sorter.
RB670 reagents have reduced monocyte background compared to PE-Cy5
Human PBMCs were stained with BD Horizon™ RB670 or PE-Cy5 Human CD8 (clone RPA-T8) Antibody, followed by erythrocyte lysis using BD Pharm Lyse™ Lysing Buffer, and acquired on a BD FACSymphony™ A5 SE Cell Analyzer and analyzed with FlowJo™ Software.
Applications
Human whole blood was stained with BD Horizon™ RB670 Reagent CD279 (EH12.1) and co-stained with BUV395 CD3 reagent (UCHT1), followed by erythrocyte lysis using BD Pharm Lyse™ Lysing Buffer and acquisition on a BD FACSymphony™ A5 SE Cell Analyzer with analysis in FlowJo™ Software.
Human PBMCs were stained with BUV395 CD4 (SK3), fixed and permeabilized using the BD Pharmingen™ Transcription Factor Buffer Set, and then stained with RB670 FoxP3 (259D/C7), followed by acquisition on a BD FACSymphony™ A5 SE Cell Analyzer and analysis using FlowJo™ Software.
BD Horizon™ RB670 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 CD3 (UCHT1, left) or CD4 (SK3, right) RB670 using several different dye lots, followed by lysis with BD FACS™ Lysing Solution. All specificities were acquired on a BD FACSymphony™ A5 SE Cell Analyzer.
Multicolor Flow Cytometry
BD Horizon™ RB670 Reagents Have Excellent Resolution as Demonstrated in an 18-color Spectral Flow Cytometry Panel Acquired on the BD FACSDiscover™ S8 Cell Sorter
BD Horizon RealYellow™ and RealBlue™ Fluorochromes work well together in an 18-color spectral human lineage panel
Human peripheral blood mononuclear cells were stained with a viability dye and antibodies against cell surface markers. Cells were analyzed on a BD FACSDiscover™ S8 Cell Sorter and data were analyzed with FlowJo™ Software v10.10. A gating strategy for detection of NK-cell subsets and representative subset phenotyping, after exclusion of dead cells and doublets, is shown.
Fluorochrome marker assignment for an 18-color spectral flow cytometry panel
| Specificity | Clone | Fluorochrome | |
|---|---|---|---|
| UV 349 nm | CD4 | SK3 | |
HLA-DR | G46-6 | ||
CD8 | RPA-T8 | ||
| Violet 405 nm | CCR7 | 2-L1-A | |
CD38 | HIT2 | ||
Live/dead | N/A | ||
CD14 | MφP9 | ||
| Blue 488 nm | CD57 | NK-1 | |
CD16 | 3G8 | ||
CD56 | B159 | ||
CD19 | SJ25C1 | ||
TCR γδ | 11F2 | ||
CD127 | A019D5 | ||
| Yellow-Green 561 nm | NKG2C | 134591 | |
CD3 | UCHT1 | ||
CD25 | 2A3 | ||
| Red 637 nm | Vδ2 TCR | 123R3 | |
CD45RA | HI100 |
Fluorochrome marker assignment for an 18-color spectral flow cytometry panel acquired on the BD FACSDiscover™ S8 Cell Sorter.
Buffer Compatibility
BD Horizon RealBlue™ 670 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 |
| 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 |
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Brochures
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Performance Guide and Chart
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.