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.
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
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.
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.
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.
Normalized emission profile of RY655 and RB670. PBMCs were stained with human CD4 (SK3) antibodies and acquired on a Cytek™ Aurora Spectral Analyzer.
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 |
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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.
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