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; it can also 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 580 |
*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 CD4 (SK3, left) or CD3 (UCHT1, 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 in a 12-color BD FACSLyric™ Cell Analyzer immune monitoring panel
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 | |
|---|---|---|---|
| Violet 405 nm | CCR7 | 2-L1-A | BV421 |
Viability | N/A | FVS510 | |
CD20 | 2H7 | BV605 | |
CD14 | MɸP9 | BV711 | |
CD3 | UCHT1 | RV828 | |
| Blue 488 nm | CD4 | SK3 | FITC |
CD8 | RPA-T8 | RB575 | |
CD16/56 | 3G8/B159 | RB705 | |
CD45RA | HI100 | RB780 | |
| Red 640 nm | 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 |
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.
For Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for Resale.
BD flow cytometers are Class 1 Laser Products.
The BD FACSLyric™ Flow Cytometer is for Research Use Only with BD FACSuite™ Application for up to 12 colors. Not for use in diagnostic or therapeutic procedures.