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.
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.