A Bright, Clean Dye for Spectral Flow Cytometry
BD Horizon RealRed™ 688 (RR688) Reagents are a bright, clean option for spectral flow cytometry panels, excited primarily by the red laser (627-640 nm) to offer:
- A bright fluorochrome to support the detection of low expression surface and intracellular markers
- Minimal cross-laser excitation
- Optimized compatibility with spectral flow cytometers
RR688 Reagents Are Optimized for Use in Spectral Flow Cytometry Panels
| Format | Ex Max | Em Max | Spectral | Conventional | Relative Brightness | Spillover* (1 = low, 4 = high) | Alternative To |
|---|---|---|---|---|---|---|---|
| RR688 | 669 nm | 688 nm | ✓ | _ | 2 | Alexa Fluor™ 660, Spark NIR™ 685, NovaFluor™ R685, cFluor® R685 |
*Value may vary based on instrument configuration and settings. Spillover ranking is based on cross-laser excitation and does not take into account spillover into adjacent detectors.
Performance
BD Horizon RealRed™ 688 Reagents Are Very Bright with Reduced Cross Laser Excitation Compared to AlexaFluor™ 660
RR688 reagents offer minimal cross-laser excitation
Normalized emission profile of CD4 (SK3) RR688, NovaFluor™ Red 685 (NFR685), Spark™ NIR 685 and CD4 (RPA-T4) AlexaFluor™ 660 (AF660). Data was acquired on a Cytek® Aurora Cell Analyzer.
RR688 reagents can be used together with APC and R718 to increase red laser utility and provide an additional option for panel design flexibility
With the addition of RR688, there are now four available positions for spectral flow cytometry using the red laser.
RR688 reagents are very bright for improved data resolution
Whole blood was stained with CD4 (SK3) RR688, NovaFluor™ R685, Spark™ NIR 685, cFluor® R685, CD4 (RPA-T4) AlexaFluor™ 660 (top row) or CD3 (UCHT1) RR688 or NovaFluor™ R685 (bottom row) and lysed with BD Pharm Lyse™ Lysing Solution. Cells were acquired and analyzed on a BD FACSymphony™ A5 SE Cell Analyzer and analyzed with FlowJo™ (v10) Software.
Applications
Human whole blood was stained with CD279 (EH12.1) RR688 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) PBMCs were stained with CD4 (SK3) BV421 and FoxP3 (259D/C7) RR699 using BD Biosciences Intracellular Cytokine Staining protocol.
B) Human whole blood was treated with BD Phosflow™ Lyse/Fix Buffer and stained with Granzyme B (GB11) RR688 and CD8 (RPA-T8) BV421. Cells were acquired and analyzed on a BD FACSymphony™ A5 SE Cell Analyzer and spectrally unmixed with FlowJo™ (v10) Software.
PBMCs were stained with human CD4 (SK3) RR688, R718 or APC and samples were acquired on a Cytek® Aurora and analyzed with FlowJo™ (v10) Software. Two-color unmixing matrices were applied and single-color overlays were generated using FlowJo™ (v10) Software.
Similarity index values were created using the BD® Spectrum Viewer tool which uses CD4 single-color stains acquired on a BD FACSDiscover™ S8 Cell Sorter.
For additional information on how these scores are calculated, please visit the BD® Spectrum Viewer.
BD Horizon™ RR688 Reagents offer 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) RR688 using four different dye lots, followed by lysis with BD Pharm Lyse™ Lysing Buffer. All specificities were run on a Cytek® Aurora Spectral Analyzer. CD3 and CD4 were tested in separate experiments.
Multicolor Flow Cytometry
BD Horizon™ RR688 Reagents Work Well in a 30-color Spectral Flow Cytometry Panel Acquired on the BD FACSDiscover™ S8 Cell Sorter
BD Horizon RealRed™, 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.
Fluorochrome marker assignment for a 30-color spectral flow cytometry panel
| Specificity | Clone | Fluorochrome | |
|---|---|---|---|
| UV 355 nm | CD2 | RPA-2.10 | |
CD8 | RPA-T8 | ||
CD14 | 63D3.rMAb | ||
CD19 | HIB19 | ||
TCR | 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 640 nm | CD56 | NCAM16.2 | |
CD20 | 2H7 | ||
TIGIT | TgMab-2 | ||
HLA-DR | G46-6 |
Fluorochrome marker assignment for a 30-color spectral flow cytometry panel run on the BD FACSDiscover™ S8 Cell Sorter.
Buffer Compatibility
BD Horizon RealRed™ 688 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.