A Bright Option for High Parameter and Spectral Flow Cytometry Panels
BD Horizon RealBlue™ 744 (RB744) Reagents leverage an innovative laser-specific fluorochrome, excited primarily by the 488-nm blue laser to offer:
- Minimal cross-laser excitation off the 561-nm yellow-green laser
- A bright fluorochrome to support the detection of low-expression surface and intracellular markers
- Compatibility with appropriately configured* conventional and spectral flow cytometers
*Filter centered near 750 nm, e.g., 750/30
RB744 Reagents Can Be Used in High Parameter Flow Cytometry Panels to Increase Biological Resolution
| Format | Ex Max | Em Max | Spectral | Conventional | Relative Brightness | Spillover* (1 = low, 4 = high) | Alternative To |
|---|---|---|---|---|---|---|---|
| RB744 | 498 nm | 746 nm | ✓ | ✓ | 1 | BB755-P |
*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 RealBlue™ 744 Reagents Provide Minimal Cross-laser Excitation Off the 561-nm Yellow-green Laser
RB744 has minimal cross-laser excitation
Normalized emission profile of RB744. Human CD4 SK3 antibody acquired on a BD FACSDiscover™ S8 Cell Sorter.
RB744 is a very bright fluorochrome with minimal spillover spread into RY743
Human whole blood was stained with BD Horizon™ RB744 Human CD4 Reagent (clone SK3), acquired on a BD FACDiscover™ S8, and unmixed with BUV737, BV750, RY743 or R718 (clone SK3) using FlowJo™ Software. Single-color overlays are shown.
Applications
Human whole blood was stained with PE (left) or BD Horizon™ RB744 Reagent (right) CD25 (2A3), co-stained with APC CD4 (SK3, data not shown) and acquired on a BD FACSymphony™ A5 SE Cell Analyzer with compensation.
Human PBMCs were stimulated with PMA and ionomycin in the presence of BD GolgiStop™ Protein Transport Inhibitor for 5 hours (top) or left unstimulated (bottom) and then stained with either PerCP-Cy5.5, BB700, or BD Horizon™ RB744 Reagent (clone B27) and matched isotype controls. Samples were acquired on a BD FACSymphony™ A5 SE Cell Analyzer and signal was analyzed using the B710 detector. Histograms are zoomed in to better show IFN-γ-positive cells.
BD Horizon™ RB744 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) RB744 using several different dye lots, followed by lysis with BD FACS™ Lysing Solution. All specificities were acquired on a BD FACSymphony™ A5 SE Cell Analyzer. BD Horizon™ and BD OptiBuild™ Reagents were tested in separate experiments.
Multicolor Flow Cytometry
BD Horizon™ RB744 Reagents Have Excellent Resolution as Demonstrated in a 17-color Spectral Flow Cytometry Panel Acquired on the BD FACSymphony™ A5 SE Cell Analyzer
Gating strategy for detection of T cell subsets in activated T cells
Peripheral blood mononuclear cells were isolated and loaded with BD Horizon™ Violet Proliferation Dye 450 before stimulation with or without staphylococcal enterotoxin B (SEB, 1 µg/mL) and CD28 (1 µg/mL) for 3 days. PMA (10 ng/mL) and ionomycin (1 µg/mL) were added to the Stimulated + Boost group 4 hours before collecting cells for staining. Cells were then stained with BD Horizon™ Fixable Viability Stain 620 and antibodies against cell surface markers prior to fixing and permeabilizing with BD Cytofix/Cytoperm™ Fixation/Permeabilization Buffer. Fixed and permeabilized cells were then stained with intracellular antibodies. Stained cells were analyzed on a BD FACSymphony™ A5 SE Cell Analyzer. Gating strategy for detection of T cell subsets after exclusion of doublets, dead cells and lineage-positive cells: CD3+ T cells were selected and divided into CD4+ or CD8+ cells. CD8+ T cells were further evaluated for activation stage based on their expression of CD25 and CD69. Samples were acquired on a BD FACSymphony™ A5 SE Cell Analyzer and analyzed with FlowJo™ Software.
Expression of inhibitory receptors at different activation levels of CD8+ T cells
Histogram overlays showing expression of inhibitory receptors on CD8+ T cell subsets from the Stimulated group. Total CD8+ T cells from the Unstimulated group (top, dark green).
Cytokines expression at different activation stages of CD8+ T cells
Histogram overlays showing expression of cytokines on CD8+ T cell subsets from the Stimulated + Boost group. Total CD8+ T cells from the Unstimulated group (top, dark green).
Fluorochrome marker assignment for a 17-color spectral flow cytometry panel
| Specificity | Clone | Fluorochrome | |
|---|---|---|---|
| UV 355 nm | CD25 | M-A251 | |
CD56 | NCAM16.2 | ||
CD20 | L27 | ||
CD19 | SJ25C1 | ||
CD16 | 3G8 | ||
CD14 | M5E2 | ||
AutoF | N/A | N/A | |
TIM-3 | 7D3 | ||
CD4 | SK3 | ||
| Violet 405 nm | TNF | MAb11 | |
VPD450 | N/A | N/A | |
CD3 | UCHT1 | ||
CD134 | ACT35 | ||
| Blue 488 nm | IFNγ | B27 | |
CD69 | FN50 | ||
FVS620 | N/A | N/A | |
LAG-3 | T47-530 | ||
PD-1 | EH12.1 | ||
II-2 | MQ1-17H12 | ||
| Yellow-Green 561 nm | CTLA-4 | BNI3 | |
| Red 640 nm | GranzB | GB11 | |
CD8 | SK1 |
Fluorochrome marker assignment for a 17-color spectral flow cytometry panel acquired on the BD FACSymphony™ A5 SE Cell Analyzer.
Buffer Compatibility
BD Horizon RealBlue™ 744 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 |
| CellBlox™ Blocking 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.