A Superior Alternative to PE-Cy7 for High-Parameter and Spectral Flow Cytometry Panels
BD Horizon RealYellow™ 775 (RY775) Reagents leverage an innovative laser-specific fluorochrome, excited primarily by the 561-nm yellow-green laser to offer:
- Reduced cross-laser excitation compared to PE-Cy7 and StarBright™ Yellow 800 (SBY800)
- A bright fluorochrome to support the detection of low-expression surface and intracellular markers
- Reduced nonspecific monocyte binding compared to PE-Cy7
RY775 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 |
|---|---|---|---|---|---|---|---|
| RY775 | 557 nm | 775 nm | ✓ | ✓ | 2 | PE-Cy7, StarBright™ Yellow 800 |
*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™ 775 Reagents Are Very Bright with Reduced Spillover and Monocyte Background
Normalized emission profiles of CD4 (SK3) RY775 and PE-Cy7, and CD4 (RPA-T4) StarBright™ Yellow 800 (SBY800) fluorochromes. Data acquired on a BD FACSDiscover™ S8 Cell Sorter.
RY775 has reduced cross-laser excitation into the corresponding UV, violet, blue and red detectors
Human PBMCs were stained with CD4 (SK3) RY775, PE-Cy7 or StarBright™ Yellow 800 (SBY800), acquired on a BD FACSDiscover™ S8 Cell Sorter and spectrally unmixed with FlowJo™ Software.
RY775 has minimal monocyte background compared to PE-Cy7
Human whole blood was stained with CD3 (UCHT1) PE-Cy7 or RY775 followed by lysis with BD FACS™ Lysing Solution. Samples run on a BD FACSymphony™ A5 SE Cell Analyzer.
Applications
Human whole blood was stained with CD279 (EH12.1) PE-Cy7 (left) or RY775 (right), co-stained with BUV395 CD3 (UCHT1) and acquired on a BD FACSymphony™ A5 SE Cell Analyzer with compensation.
BD Pharmingen™ HiCK-1 Human Cytokine Positive Control Cells (top) or unstimulated human PBMC (bottom) were permeabilized and then stained with either PE-Cy7 or RY775 IFN-γ (B27) and matched isotype controls. Samples were acquired on a BD FACSymphony™ A5 SE Cell Analyzer.
BD Horizon™ RY775 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) RY775 using several different dye lots, followed by lysis with BD FACS™ Lysing Solution. All samples were acquired on a BD FACSymphony™ A5 SE Cell Analyzer. BD Horizon™ and BD OptiBuild™ Reagents were tested in separate experiments.
Multicolor Flow Cytometry
RY775 Reagents Have Excellent Resolution as Demonstrated in an 18-color Spectral Flow Cytometry Panel Acquired on the BD FACSDiscover™ S8 Cell Sorter
RY775 and BD Horizon RealBlue™ 780 (RB780) Reagents work well together in an 18-color spectral human immunophenotyping 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 B cells and NK and T-cell subsets, 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 | ||
CD19 | SJ25C1 | ||
TCR γδ | 11F2 | ||
CD127 | A019D5 | ||
| Yellow-Green 561 nm | NKG2C | 134591 | |
CD3 | UCHT1 | ||
CD25 | 2A3 | ||
CD56 | B159 | ||
| 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 RealYellow™ 775 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 |
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.