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A Bright Option for High Parameter and Spectral Flow Cytometry Panels
BD Horizon RealYellow™ 655 (RY655) Reagents leverage an innovative laser-specific fluorochrome, excited primarily by the 561-nm yellow-green laser to offer:
- Limited cross-laser excitation from the 488-nm blue laser
- A bright fluorochrome to support the detection of low-expression surface and intracellular markers
- Reduced nonspecific monocyte binding compared to legacy PE tandem fluorochromes
- Compatible with appropriately configured* conventional and spectral flow cytometers
*Filter centered near 660 nm e.g., 660/30 or 670/30. Note that a 670/30 configuration on a conventional instrument may not capture the max signal and thus higher antigen density markers should be assigned.
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RY655 Reagents Can Be Used in Higher Parameter Flow Cytometry Panels to Increase Biological Resolution
| Format | Ex Max | Em Max | Spectral | Conventional | Relative Brightness | Spillover* (1 = low, 4 = high) | Alternative To |
|---|---|---|---|---|---|---|---|
| RY655 | 557 nm | 654 nm | ✓ | ✓ | 3 | PE-Cy5, StarBright™ Yellow 665 |
*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™ 655 Reagents Are Very Bright with Reduced Spillover and Monocyte Background
RY655 has reduced cross-laser excitation compared to PE-Cy5
Normalized emission profile of CD4 (SK3) RY655, PE-Cy5, NovaFluor™ Yellow 660 (NFY660) and PE/Fire™ 640, and CD4 (RPA-T4) StarBright™ Yellow 665 (SBY665). Data was acquired on a BD FACSDiscover™ S8 Cell Sorter.
RY655 has reduced spread into RB670 compared to PE-Cy5
PBMCs were stained with CD4 (SK3) RY655, RB670 or PE-Cy5. Cells were acquired and analyzed on a Cytek™ Aurora Spectral Analyzer and unmixed with FlowJo™ (v10) Software. Single-color stains are overlaid.
RY655 has minimal monocyte background compared to PE-Cy5
Human whole blood was stained with Mouse IgG1, κ Isotype Control (X40 for RY655 and MOPC21 for PE-Cy5), CD3 (UCHT1), CD8 (RPA-T8) or CD16 (3G8) PE-Cy5 or RY655 followed by lysis with BD Pharm Lyse™ Lysing Solution. Samples run on a BD FACSymphony™ A5 SE Cell Analyzer.
Applications
BD Horizon™ RY655 Reagents resolve low-expression surface markers
Human whole blood was stained with CD279 (EH12.1) RY655 and CD3 (UCHT1) FITC followed by lysis with BD Pharm Lyse™ Lysing Solution, acquired on a BD FACSymphony™ A5 SE Cell Analyzer and spectrally unmixed with FlowJo™ (v10) Software.
BD Horizon™ RY655 Reagents resolve intracellular markers
A) Stimulated PBMCs stained with BD Horizon™ BUV395 CD3 (UCHT1) and BD Horizon™ RY655 IFN-γ (B27) using the Immunofluorescent Staining of Intracellular Cytokines for Flow Cytometric Analysis protocol
B) Human whole blood was treated with BD Phosflow™ Lyse/Fix Buffer and stained with BD Horizon™ RY655 Granzyme B (GB11) and BD Horizon™ BV421 CD8 (RPA-T8). Cells were acquired and analyzed on a BD FACSymphony™ A5 SE Cell Analyzer and spectrally unmixed with FlowJo™ (v10) Software.
RY655 can be used with other fluorochromes excited by the 561-nm yellow-green laser and with its blue laser pair, RB670
Human whole blood was stained with anti-CD4 fluorochromes followed by lysis with BD Pharm Lyse™ Lysing Buffer and similarity scores were calculated. Single-color stains were acquired on a BD FACSymphony™ A5 SE Cell Analyzer and analyzed with FlowJo™ (v10) Software.
RY655 can be used with RB670 on instruments with both blue and yellow-green lasers and the appropriate detectors
Normalized emission profile of RY655 and RB670. PBMCs were stained with human CD4 (SK3) antibodies and acquired on a Cytek™ Aurora Spectral Analyzer.
RY655 lot-to-lot consistency is demonstrated across made-to-stock and BD OptiBuild™ On-Demand Reagents
BD Horizon™ RY655 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) RY655 using three different dye lots, followed by lysis with BD Pharm Lyse™ Lysing Buffer. All samples were acquired on a Cytek™ Aurora Spectral Analyzer. CD3 and CD4 were tested in separate experiments.
Multicolor Flow Cytometry
BD Horizon™ RY655 Reagents Have Excellent Resolution as Demonstrated in a 30-color Spectral Flow Cytometry Panel Acquired on the BD FACSDiscover™ S8 Cell Sorter
BD Horizon RealYellow™ and RealBlue™ Reagents work well together in a 30-color spectral human immunophenotyping panel
Human peripheral blood mononuclear cells were stained with a viability dye and antibodies against cell surface markers, using BD Horizon™ Brilliant Stain Buffer Plus. Cells were analyzed on a BD FACSDiscover™ S8 Cell Sorter and data were analyzed with FlowJo™ (v10) Software. A gating strategy for immunophenotyping, including detection of dendritic cells, monocytes, B-cells, NK-cell subsets and T-cell subsets, after exclusion of dead cells and doublets, is shown. CD95 RY655 enables effective identification of the rare plasmablast cell population within gate 17.
Fluorochrome marker assignment for a 30-color spectral flow cytometry panel
| Specificity | Clone | Fluorochrome | |
|---|---|---|---|
| UV 349 nm | CD2 | RPA-2.10 | |
CD8 | RPA-T8 | ||
CD14 | 63D3.rMAb | ||
CD19 | HIB19 | ||
TCRγδ | 11F2 | ||
CD45 | HI30 | ||
| Violet 405 nm | CCR7 | 2-L1-A | |
CD45RA | HI100 | ||
Viability | N/A | ||
CD38 | HB7 | ||
CD123 | 9F5 | ||
CD161 | DX12 | ||
CD16 | 3G8 | ||
| Blue 488 nm | CD3 | UCHT1 | |
CD57 | NK-1 | ||
CD25 | 2A3 | ||
CX3CR1 | 2A9-1 | ||
PD-1 | EH12.1 | ||
CD127 | HIL-7R-M21 | ||
CD28 | CD28.2 | ||
| Yellow-Green 561 nm | KLRG1 | Z7-205.rMAb | |
CD4 | SK3 | ||
CD95 | DX2 | ||
TIM-3 | 7D3 | ||
CD11c | B-ly6 | ||
CD27 | M-T271 | ||
| Red 637 nm | CD56 | NCAM16.2 | |
CD20 | 2H7 | ||
TIGIT | TgMab-2 | ||
HLA-DR | G46-6 |
Fluorochrome marker assignment for a 30-color spectral flow cytometry panel acquired on the BD FACSDiscover™ S8 Cell Sorter.
Buffer Compatibility
BD Horizon RealYellow™ 655 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 |
RESOURCES
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Brochures
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Poster
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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.