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
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.