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.
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
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.