A Moderately Bright Fluorochrome with Narrow Emission off the Violet Laser
BD Horizon RealViolet™ 544 (RV544) Reagents leverage an innovative laser-specific fluorochrome, excited primarily by the 405-nm violet laser to offer:
- Narrow emission off the violet laser
- Moderate brightness to support the detection of medium- to high-expression surface and intracellular markers
- Compatibility with neighboring BD Horizon Brilliant Violet™ Fluorochromes, enabling use in high parameter spectral panels
RV544 Reagents Can Be Used with Moderate- to High-Density Antigens on Spectral Flow Cytometers
| Format | Ex Max | Em Max | Spectral | Conventional | Relative Brightness | Spillover* (1 = low, 4 = high) | Alternative To |
|---|---|---|---|---|---|---|---|
| RV544 | 412 nm | 546 nm | ✓ | - | 2 | Spark™ V538, StarBright™ Violet 535, Pacific Orange, Amethyst Orange, cFluor™ V547, Krome Orange |
*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 RealViolet™ 544 Reagents Provide Narrow Emission Off the 405-nm Violet Laser and Reduced Cross-laser Excitation Off the 355-nm UV Laser
RV544 has narrow emission off the violet laser and reduced UV-cross-laser excitation
Normalized emission profiles of RV544, Spark Violet™ 538 (Spark V538) and Pacific Orange. PBMCs stained with human CD4 SK3 antibody (RV544 and Spark V538) or human CD4 RPA-T4 (Pacific Orange) and acquired on a BD FACSDiscover™ S8 Cell Sorter.
RV544 works well with neighboring BD Horizon Brilliant Violet™ Fluorochromes
Spectral signature heat map of RV544 (pink) with all BD Horizon Brilliant™ Violet Fluorochromes and BD Horizon RealViolet™ 828 (RV828) Fluorochrome. 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™ RV544 Mouse IgG2b Isotype or BD Horizon™ RV544 Mouse Anti-Human CD20 Antibody. The erythrocytes were lysed with BD FACS™ Lysing Solution. Samples were acquired on the BD FACSymphony™ A5 SE Cell Analyzer and spectrally unmixed with autofluorescence using FlowJo™ Software.
Human whole blood (Granzyme B and matched isotype) or HICK1 cells (IFN-γ and matched isotype) were fixed and permeabilized, then stained with either human CD8 BUV395 and Granzyme B RV544 or human CD3 BUV395 and IFN-γ RV544. Samples were acquired on the BD FACSymphony™ A5 SE Cell Analyzer and spectrally unmixed with autofluorescence using FlowJo™ Software.
PBMCs were stained with either BD Horizon™ RV544 or Spark Violet™ 538 (Spark V538) human CD4 (SK3) followed by fixation and permeabilization with BD Phosflow™ Perm Buffer III. Cells were acquired on a BD FACSymphony™ A5 SE Cell Analyzer and analyzed with FlowJo™ Software.
BD Horizon™ RV544 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) RV544 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™ RV544 works well for spectral flow cytometry panels
RV544 works well with neighboring fluorochromes on a spectral cell sorter
Similarity index values from the BD FACSDiscover™ S8 Cell Sorter. RV544 is compatible with existing neighboring and cross-laser dyes.
RV544 works well in a 31-color spectral human immunophenotyping BD FACSDiscover™ A8 Cell Analyzer panel
Human peripheral blood mononuclear cells were stained with a viability dye and antibodies against cell surface markers. BD Horizon™ Brilliant Stain Buffer Plus was used to mitigate dye-dye interactions. Cells were analyzed on a BD FACSDiscover™ A8 Cell Analyzer and data were analyzed with FlowJo™ Software v10.10. A gating strategy for immunophenotyping, including detection of dendritic cells, monocytes, B cells, NK cell subsets and T cell subsets, after exclusion of dead cells and doublets, is shown.
Fluorochrome marker assignment for a 31-color human immune monitoring panel
| Specificity | Clone | Fluorochrome | |
|---|---|---|---|
| UV 349 nm | CD45RA | HI100 | BUV395 |
| CD8 | RPA-T8 | BUV496 | |
| CD38 | HIT2 | BUV615 | |
| CD19 | HIB19 | BUV661 | |
| TCR γδ | 11F2 | BUV737 | |
| CD185 (CXCR5) | RF8B2 | BUV805 | |
| Violet 405 nm | CD45 | HI30 | V450 |
CD197 (CCR7) | 2-L1-A | BV421 | |
CD14 | M5E2 | BV480 | |
CD3 | UCHT1 | RV544 | |
Viability | N/A | FVS575V | |
| CD24 | ML4 | BV786 | |
| CD11c | B-Ly6 | RV828 | |
| Blue 488 nm | CD57 | NK-1 | FITC |
CD20 | 2H7 | RB545 | |
CD16 | 3G8 | RB575 | |
CD25 | 2A3 | RB613 | |
| CX3CR1 | 2A9-1 | RB670 | |
| CD279 (PD-1) | EH12.1 | RB705 | |
| CD127 (IL-7Rα) | HIL-7R-M21 | RB744 | |
| CD28 | CD28.2 | RB780 | |
| CD4 | SK3 | RB824 | |
| Yellow-Green 561 nm | KLRG1 | Z7-205.rMAb | RY586 |
CD56 | B159 | RY610 | |
CD95 (Fas) | DX2 | RY655 | |
| CD366 (TIM-3) | 7D3 | RY703 | |
| CD272 (BTLA) | J168-540 | RY743 | |
| CD27 | M-T271 | RY775 | |
| Red 637 nm | CD161 | DX12 | RR688 |
| TIGIT | TgMab-2 | R718 | |
| HLA-DR | G46-6 | APC-H7 |
Buffer Compatibility
BD Horizon™ RV544 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 |
Other BD Horizon RealViolet™ and BD Horizon Fluorochromes
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 sheets before using this product as described. Comparisons, where applicable, are made against older BD technology, manual methods or are general performance claims. Comparison are not made against non-BD technologies, unless otherwise noted.