A Bright, Clean Dye
BD Horizon RealYellow™ 743 (RY743) 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
- Compatibility with spectral and appropriately configured* conventional flow cytometers
*Filter centered near 750 nm, e.g., 750/30
RY743 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 |
|---|---|---|---|---|---|---|---|
| RY743 | 557 nm | 743 nm | ✓ | ✓ | 2 | PE/Fire™ 744, NovaFluor™ Yellow 755 |
*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™ 743 Reagents Are Very Bright with Reduced Spillover and Monocyte Background
RY743 has greatly reduced cross-laser excitation
Normalized emission profiles of CD4 (SK3) RY743, PE/Fire™ 744 and NovaFluor™ Yellow 755 (NFY755) Fluorochromes. Human CD4 (SK3) antibody acquired on a BD FACSDiscover™ S8 Cell Sorter.
RY743 has reduced spread into RB744 compared to PE/Fire™ 744
PBMCs were stained with CD4 RY743, NFY755, PE/Fire™ 744 or RB744. Cells were acquired and analyzed on a Cytek™ Aurora Spectral Analyzer and unmixed with FlowJo™ Software. Single-color stains are overlaid.
RY743 has minimal monocyte background compared to PE-Cy7
Human whole blood was stained with Mouse IgG1, K Isotype Control (X40), CD3 (UCHT1), CD8 (RPA-T8) or CD19 (SJ25C1) PE-Cy7 or RY743 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) RY743 and CD3 FITC acquired on a BD FACSymphony™ A5 SE Cell Analyzer and spectrally unmixed with FlowJo™ (v10) Software.
A) Stimulated BALB/c Mouse splenic leucocytes were stained with BD Horizon™ BUV395 Rat Anti-Mouse CD4 and BD Horizon™ RY743 Rat Anti-Mouse IFN-γ using the Immunofluorescent Staining of Intracellular Cytokines for Flow Cytometric Analysis protocol.
B) Human PBMCs were stained with FoxP3 RY743 and CD4 BUV395 (left) or stimulated PBMCs were stained with IL-2 RY743 and CD3 BUV395 (right). Cells were acquired and analyzed on a BD FACSymphony™ A5 SE Cell Analyzer and spectrally unmixed with FlowJo™ Software.
PBMCs were stained with anti-CD4 fluorochromes and similarity indices were calculated. Single-color stains were acquired on a BD FACSymphony™ A5 SE Cell Analyzer and analyzed with FlowJo™ Software.
A) Normalized emission profile of RY743 and RB744. Human CD4 (clone SK3) antibodies acquired on a BD FACSDiscover™ S8 Cell Sorter.
B) Lysed whole blood was stained with either CD3 (left) or CD4 (right) RY743 and CD19 RB744. Cells were acquired and analyzed on a BD FACSymphony™ A5 SE Cell Analyzer and compensated with FlowJo™ Software.
Normalized emission profile of RY703, RY743 and RY775. Human CD4 (clone SK3) antibodies acquired on a BD FACSDiscover™ S8 Cell Sorter.
BD Horizon™ RY743 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) RY743 using three different dye lots, followed by lysis with BD Pharm Lyse™ Lysing Solution. All samples were acquired on a Cytek™ Aurora Spectral Analyzer. CD3 and CD4 were tested in separate experiments.
Multicolor Flow Cytometry
BD Horizon™ RY743 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™ Reagents work well together in an 18-color spectral murine immunophenotyping panel
BALB/c mouse spleen cells or lymph nodes and Peyer’s patches were stained with a viability dye and antibodies against cell surface markers. Brilliant Stain Buffer was used to mitigate dye-to-dye interactions. Cells were then fixed and permeabilized using the BD Pharmingen™ Transcription Factor Buffer Set and stained with antibodies against transcription factors. Cells were analyzed on a BD FACSDiscover™ S8 Cell Sorter and data were analyzed with FlowJo™ (v10) Software. A gating strategy for detection and phenotyping of various cell types, including T follicular helper cells (Tfh), T follicular regulatory cells (Tfr), Tregs and germinal center (GC) B-cells, 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 | CD3 | 145-2C11 | |
| Violet 405 nm | FoxP3 | MF23 | |
CD62L | MEL-14 | ||
Live/dead | N/A | ||
IgD | 11-26c.2a | ||
| Blue 488 nm | iCOS | 7E.17G9 | |
CD4 | GK1.5 | ||
CXCR5 | 2G8 | ||
GL-7 | GL7 | ||
CD95 | Jo2 | ||
BCL16 | K112-91 | ||
| Yellow-Green 561 nm | PD-1 | RMP1-30 | |
CD44 | IM7 | ||
CD11b | M1/70 | ||
Helios | 22F6 | ||
B220 | RA3-6B2 | ||
| Red 637 nm | CD8a | 53-6.7 | |
CD25 | 7D4 |
Fluorochrome marker assignment for an 18-color spectral flow cytometry panel acquired on the BD FACSDiscover™ S8 Cell Sorter.
Buffer Compatibility
BD Horizon RealYellow™ 743 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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