A Moderately Bright Fluorochrome with Minimal Cross-laser Excitation
BD Horizon RealBlue™ 575 (RB575) Reagents leverage an innovative laser-specific fluorochrome, excited primarily by the 488-nm blue laser to offer:
- Minimal cross-laser excitation
- Moderate brightness to support the detection of moderate- to high-expression surface and intracellular markers
- A moderate-brightness alternative to PE for configurations without a yellow-green laser, or to be used with BD Horizon RealYellow™ 586 (RY586) Reagents on appropriately configured instruments with a yellow-green laser
RB575 Reagents Can Be Used with Moderate- to High-Antigen-Expression Markers on Appropriately Configured Conventional and Spectral Flow Cytometers
| Format | Ex Max | Em Max | Spectral | Conventional | Relative Brightness | Spillover* (1 = low, 4 = high) | Alternative To |
|---|---|---|---|---|---|---|---|
| RB575 | 508 nm | 574 nm | ✓ | _ | 1 | PE, StarBright™ Blue 580cFluor® R685 |
*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™ 575 Reagents Provide Minimal Cross-laser Excitation Off the 561-nm Yellow-green Laser
RB575 reagents have reduced cross-laser excitation compared to PE
Normalized emission profiles of RB575 and PE. PBMCs stained with human CD4 SK3 antibody and acquired on a BD FACSDiscover™ S8 Cell Sorter.
RB575 and RY586 are excited by their primary lasers and pair well together on instruments with both blue and yellow-green lasers
Normalized emission profiles of RB575 and RY586. PBMCs stained with human CD4 SK3 antibody and acquired on a BD FACSDiscover™ S8 Cell Sorter.
Applications
Human whole blood was stained with either BD Horizon™ RB575 Mouse IgG1, κ Isotype Control or BD Horizon™ RB575 Mouse Anti-Human CD28 Antibody. The erythrocytes were lysed with BD Pharm Lyse™ Lysing Buffer. Samples were acquired on the BD FACSymphony™ A5 SE Cell Analyzer and spectrally unmixed with autofluorescence using FlowJo™ Software.
Stimulated PBMCs were stained with IFN-γ RB824 and CD3 BUV395. Samples were acquired on a BD FACSymphony™ A5 SE Cell Analyzer and spectrally unmixed with FlowJo™ Software.
Human whole blood was stained with either RB575, StarBright™ Blue 580 (SBB580), PE or PE-Cy7 CD19 (clone SJ25C1 for all except SBB580, which is LT19) and lysed with BD Pharm Lyse™ Lysing Buffer. Cells were acquired on a BD FACSymphony™ A5 SE Cell Analyzer and analyzed with FlowJo™ Software. PE and PE-Cy7 were analyzed in the peak detector off the blue laser, BP-B576 and BP-B810 respectively.
A) PBMCs were stained with BD Horizon™ RB575 or PE human CD3 (UCHT1) and then fixed and permeabilized with BD Phosflow™ Perm Buffer III. B) Whole blood was stained with BD Horizon™ RB575 or PE Human CD4 (SK3) and lysed and fixed with BD FACS™ Lysing Solution, followed by permeabilization with BD FACS™ Permeabilizing Solution 2.
BD Horizon™ RB575 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) RB575 using three different dye lots, followed by lysis with BD Pharm Lyse™ Lysing Buffer. All specificities were run on a Cytek™ Aurora Spectral Analyzer.
Multicolor Flow Cytometry
BD Horizon™ RB575 works well for both conventional and spectral flow cytometry panels
RB575 works well with neighboring fluorochromes on a spectral cell sorter
Similarity index values from the BD FACSDiscover™ S8 Cell Sorter. RB575 is compatible with existing neighboring and cross-laser dyes. RB575 has a similarity index of 0.95 or above with NovaFluor™ B585 (NF B585) and Spark Blue™ 575 (Spark B574), so running them in the same panel is not recommended.
RB575 works well with neighboring fluorochromes on a spectral cell sorter
Whole blood was bulk lysed with BD Pharm Lyse™ Lysing Buffer, washed and stained with BD Horizon™ Fixable Viability Stain 510, prior to being stained with other surface markers. Cells were fixed with 4% PFA, acquired on a BD FACSLyric™ Cell Analyzer and analyzed with FlowJo™ Software.
Fluorochrome marker assignment for a 12-color human immune monitoring panel
| Specificity | Clone | Fluorochrome |
|---|---|---|
CCR7 | 2-L1-A | BV421 |
Viability | N/A | FVS510 |
CD20 | 2H7 | BV605 |
CD14 | MɸP9 | BV711 |
CD3 | UCHT1 | RV828 |
CD4 | SK3 | FITC |
CD8 | RPA-T8 | RB575 |
CD16/56 | 3G8/B159 | RB705 |
CD45RA | HI100 | RB780 |
CD19 | SJ25C1 | APC |
CD95 | DX2 | R718 |
CD45 | 2D1 | APC-H7 |
Buffer Compatibility
BD Horizon™ RB575 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.