Violet-laser-excited NIR Fluorochrome with Minimal Cross-laser Excitation
BD Horizon RealViolet™ 828 (RV828) Reagents leverage an innovative laser-specific fluorochrome, excited primarily by the 405-nm violet laser to offer:
- Minimal cross-laser excitation
- A moderate-brightness fluorochrome to support the detection of moderate to high expression surface and intracellular markers
- A near-infrared (NIR) detection position for the violet laser
RV828 Delivers Robust Detection of Moderate to High Expression Markers, Optimized for Peak Performance on Spectral Flow Cytometers
| Format | Ex Max | Em Max | Spectral | Conventional | Relative Brightness | Spillover* (1 = low, 4 = high) | Alternative To |
|---|---|---|---|---|---|---|---|
| RV828 | 412 nm | 828 nm | ✓ | - | 1 | Qdot™ 800, NovaFluor™ V800 |
*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™ 828 Reagents Deliver Minimal Cross-laser Excitation
RV828 has minimal cross-laser excitation
Normalized emission profiles of CD4 (SK3) RV828 and NovaFluor™ V800 (NFV800), and CD4 (S3.5) Qdot™ 800. Data acquired on a BD FACSDiscover™ S8 Cell Sorter.
RV828 has less spread compared to Qdot™ 800
PBMCs were stained with the following reagents: CD4 RV828, Qdot™ 800, BUV805, RB824, SBY800 or APC/Fire™ 810. Shown are single color overlays highlighting RV828 and Qdot™ 800 cross-laser spread. Samples were acquired on a Cytek™ Aurora Spectral Analyzer and unmixed using FlowJo™ Software.
Applications
Whole blood was stained with CD28 RV828 and lysed with BD Pharm Lyse™ Lysing Buffer. Samples were acquired on a BD FACSymphony™ A5 SE Cell Analyzer and spectrally unmixed with lymphocyte autofluorescence using FlowJo™ Software.
BALB/c mouse thymocytes were stained with CD4 APC and then fixed and permeabilized using the BD Pharmingen™ Transcription Factor Buffer Set. The cells were then stained with either RV828 Isotype Control or RV828 Mouse Anti-Mouse RORγt antibody. The overlapping histograms show the levels of either Isotype Control (dashed line) or RORγt (solid line) staining in CD4+ thymocytes. Samples were acquired on the BD FACSymphony™ A5 SE Cell Analyzer and spectrally unmixed using FlowJo™ Software.
PBMCs were stained with either BD Horizon™ RV828 or NovaFluor™ V800 Human CD4 (SK3) either before or after they were fixed with 2% PFA and permeabilized using BD Phosflow™ Perm III Buffer. Samples were acquired on a Cytek™ Aurora and analyzed with FlowJo™ Software.
BD Horizon™ RV828 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) RV828 using three different dye lots, followed by lysis with BD Pharm Lyse™ Lysing Buffer. All specificities were run on a Cytek™ Aurora Spectral Analyzer. CD3 and CD4 were tested in separate experiments.
Multicolor Flow Cytometry
BD Horizon™ RV828 Reagents Resolve Well in a 24-color T Cell Activation Panel Acquired on the BD FACSDiscover™ A8 Cell Analyzer
Gating scheme of T cell subsets
PBMCs were isolated from a healthy donor and divided into two conditions: unstimulated and stimulated with CytoStim™ (Miltenyi Biotec). After 24 hours, both samples were collected and labeled with CD45. CAR T cells were spiked into the stimulated sample. Samples were then stained with the remaining 23 markers of the 24-color T cell activation panel incorporating BD Horizon RealViolet™ 828 and BD Horizon RealBlue™ 824 (RB824), along with a BD® CD19 CAR Detection Reagent, and analyzed on the BD FACSDiscover™ A8 Cell Analyzer.
A) Gating strategy for T cell phenotyping of unstimulated PBMC.
B) Pseudocolor plots show detection of CD19 CAR+ T cells in the stimulated PBMC sample mixed with CAR T cells. Contour plots show expression of memory, activation and inhibitory markers in unstimulated PBMC, stimulated PBMC and CD19 CAR+ T cells.
Fluorochrome marker assignment for a 24-color human T cell activation panel
| Specificity | Clone | Fluorochrome | |
|---|---|---|---|
| UV 349 nm | CD8 | RPA-T8 | |
CD45 | HI30 | ||
CD25 | 2A3 | ||
| Violet 405 nm | CCR7 | 2-L1-A | |
CD45RA | HI100 | ||
Viability | N/A | ||
CD20 | 2H7 | ||
CD3 | UCHT1 | ||
| Blue 488 nm | CD28 | CD28.2 | |
CD95 | DX2 | ||
CCR6 | 11A9 | ||
CXCR3 | 1C6/CXCR3 | ||
CD127 | HIL-7R-M21 | ||
KLRG1 | Z7-205.rMAb | ||
CD45RO | UCHL1 | ||
| Yellow-Green 561 nm | TIM-3 | 7D3 | |
CD69 | FN50 | ||
TCRγδ | 11F2 | ||
CD56 | B159 | ||
CD16 | 3G8 | ||
CCR4 | 1G1 | ||
| Red 637 nm | CD19 | N/A | |
PD-1 | EH12.1 | ||
TIGIT | TgMab-2 | ||
CD4 | RPA-T4 |
Fluorochrome marker assignment for a 24-color spectral flow cytometry panel acquired on the BD FACSDiscover™ A8 Cell Analyzer.
Buffer Compatibility
BD Horizon™ RV828 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 |
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Poster
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Performance Guide and Chart
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.