A Bright Option for High Parameter and Spectral Flow Cytometry Panels
BD Horizon RealBlue™ 613 (RB613) Reagents leverage an innovative laser-specific fluorochrome, excited primarily by the 488-nm blue laser to offer:
- A bright fluorochrome to support the detection of low-expression surface and intracellular markers
- Significantly reduced cross-laser excitation off the 561-nm yellow-green laser
- Compatibility with conventional and spectral flow cytometers with the appropriate detectors
RB613 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 |
|---|---|---|---|---|---|---|---|
| RB613 | 492 nm | 613 nm | ✓ | ✓ | 2 | PE-CF594, PE-Dazzle™ 594, StarBright™ Blue 615, NovaFluor™ Blue 610, cFluor™ BYG610, BB630-P2 |
*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™ 613 Reagents Are the Best Choice in This Position for Flow Cytometers With a 488-nm Blue Laser
Normalized emission profiles of RB613 and PE-CF594 fluorochromes. Human CD4 SK3 antibody acquired on a BD FACSDiscover™ S8 Cell Sorter.
RB613 reagents have reduced cross-laser excitation
Human PBMCs were stained with BD Horizon™ RB613 Human CD4 (clone SK3) Antibody, acquired on a BD FACSDiscover™ S8 Cell Sorter and unmixed with FlowJo™ Software.
Applications
Human whole blood was stained with BD Horizon™ PE-CF594 or BD Horizon™ RB613 Reagent CD279 (EH12.1) and costained with APC CD3 reagent (UCHT1) followed by acquisition on a BD FACSymphony™ A5 SE Cell Analyzer with compensation in FlowJo™ Software.
Human PBMCs were fixed and permeabilized using the BD Pharmingen™ Transcription Factor Buffer Set. Cells were then stained with PE-CF594 (left) or RB613 (right) FoxP3 (259D/C7) and costained with APC CD4 (SK3) followed by acquisition on a BD FACSymphony™ A5 SE Cell Analyzer and compensation with FlowJo™ Software.
BD Horizon™ RB613 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) RB613 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
BD Horizon™ RB613 Reagents Have Excellent Resolution as Demonstrated in a 22-color Spectral Flow Cytometry Panel Acquired on the BD FACSDiscover™ S8 Cell Sorter
RB613 and RY610 reagents work well together in a 22-color spectral human lineage panel
Human peripheral fresh whole blood samples were stained with antibodies against cell surface markers prior to bulk red blood cell lysis with BD Pharm Lyse™ Lysing Buffer. Cells were then analyzed on a BD FACSDiscover™ S8 Cell Sorter and analyzed with FlowJo™ Software v10.9. A gating strategy for detection of NK and T cell subsets, after exclusion of doublets and B cells, is shown.
Fluorochrome marker assignment for a 22-color spectral flow cytometry panel
| Specificity | Clone | Fluorochrome | |
|---|---|---|---|
| UV 349 nm | CD56 | R19-760 | |
AutoF | N/A | N/A | |
CD20 | L27 | ||
CD14 | M5E2 | ||
CD127 | HIL-7R-M21 | ||
IgD | IA6-2 | ||
| Violet 405 nm | IgM | G20-127 | |
CD38 | HIT2 | ||
CD8 | RPA-T8 | ||
IgG | G18-145 | ||
CD16 | 3G8 | ||
CD19 | SJ25C1 | ||
| Blue 488 nm | CD45 | HI30 | |
CD24 | ML5 | ||
CD25 | 2A3 | ||
CD123 | 7G3 | ||
TCRgd | 11F2 | ||
BAFF-R (CD268) | 11C1 | ||
| Yellow-Green 561 nm | CD10 | HI10a | |
CD3 | UCHT1 | ||
| Red 637 nm | CD4 | SK3 | |
CD27 | M-T271 | ||
HLA-DR | L243 |
Fluorochrome marker assignment for a 22-color spectral flow cytometry panel acquired on the BD FACSDiscover™ S8 Cell Sorter.
Buffer Compatibility
BD Horizon RealBlue™ 613 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 |
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Refer to manufacturer's instructions for use and related User Manuals and Technical Data Sheets before using this product as described.
Comparisons, where applicable, are made against older BD technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
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.