Multicolor Flow Cytometry

Brilliant Violet™ 421 offers a violet-excitable dye with significantly improved brightness over other dyes offered for the violet laser. Additionally, Brilliant Violet reagents are brighter than reagents for the blue and red lasers, making Brilliant Violet 421 the brightest dye offered by BD. The brightness of the dye makes it particularly useful in multicolor applications where it can be used to better resolve dim populations.

A bright dye for the violet laser

Brilliant Violet 421 is a polymer-based dye developed by Sirigen from Nobel Prize winning chemistry that provides the high sensitivity fluorescence needed to resolve dim populations. This polymer technology combines the ease of use of a traditional organic dye with the brightness of phycoerythrin (PE) for the violet laser. Brilliant Violet 421 conjugates are, on average, 10 times brighter than Pacific Blue™ conjugates and are often 2–3 times brighter than PE conjugates (Table 1, Figure 2). Brilliant Violet 421 is optimized for use on BD FACS™ brand flow cytometers equipped with a violet laser, including the BD FACSVerse™ flow cytometer, the BD™ LSR cell analyzer platform, the BD FACSAria™ cell sorter platform, and the BD FACSCanto™ II flow cytometer. With maximum emission peaks at 421 nm and 448 nm, Brilliant Violet 421 is compatible with the standard BD Horizon™ V450 filter set (eg, 450/50 nm) (Figure 1).

Table 1. Stain index comparison

Table 1. Stain index comparison

Lysed whole blood stained with CD4, CD8, or CD127 conjugated to Brilliant Violet 421, Pacific Blue™, BD Horizon V450, VioBlue®, or PE, run on a BD LSRFortessa™ flow cytometer. All conjugates were run at optimal concentration. Data was gated on lymphocytes.

Figure 1. Absorption and emission spectra

Figure 1. Absorption and emission spectra

Ex Max: 407 nm, Em Max: 421 nm and 448 nm

Figure 2. Staining comparison of Brilliant Violet 421, Pacific Blue™, BD Horizon V450, VioBlue®, and PE.

Figure 2. Staining comparison of Brilliant Violet 421, Pacific Blue™, BD Horizon V450, VioBlue®, and PE.

1. CD4: Lysed whole blood stained with human CD4 conjugated to Brilliant Violet 421, Pacific Blue™, VioBlue®, BD Horizon V450, or PE, run on a BD LSRFortessa flow cytometer. All conjugates were run at the manufacturer's recommended concentration.
2. CD8: Lysed whole blood stained with human CD8 conjugated to Brilliant Violet 421, Pacific Blue™, or PE, run on a BD LSRFortessa flow cytometer. All conjugates were run at the manufacturer's recommended concentration.

Excellent population resolution

In many cases, the typical violet excitable dyes are not bright enough to adequately resolve dim populations when compared to existing bright fluorochromes such as PE or Alexa Fluor® 647. However, as shown above, the Brilliant Violet 421 polymer is a very bright fluorochrome that provides excellent population resolution when coupled to antibodies directed at low antigen density markers. This fluorochrome brightness, in conjunction with the low spillover of other fluorchromes into it, contributes to the Brilliant Violet 421 conjugates providing equal or superior resolution over dyes excited by the blue and red lasers.

The following example compares the staining of CD25 and CD127 labeled with Brilliant Violet 421 to that of PE and Alexa Fluor® 647 respectively, in a 7-color panel used to identify regulatory T cells. Two different panels were run in parallel: one contained CD25 Brilliant Violet 421 and CD127 Alexa Fluor® 647 (Figures 3a and 3d) and the other contained CD25 PE and CD127 Brilliant Violet 421 (Figures 3b and 3c). The following dot plots demonstrate how CD25 Brilliant Violet 421 resulted in complete separation between the CD25 negative and positive populations compared to the PE conjugate. Superior separation is also seen with CD127 Brilliant Violet 421 compared to CD127 Alexa Fluor® 647. The brightness of these two Brilliant Violet 421 conjugates allows for easy and unequivocal identification of the CD25^bright CD127^dim population (regulatory T cells) as shown in Figures 3e-f.

Figure 3. Comparison data from two regulatory T-cell panels.

Figure 3. Comparison data from two regulatory T-cell panels.

Figures 3a and 3b compare the brightness of CD25 Brilliant Violet 421 with CD25 PE. Figures 3c and 3d compare the brightness of CD127 Brilliant Violet 421 with CD127 Alexa Fluor® 647.

Figures 3e and f show how the Brilliant Violet 421 reagents contribute to the unequivocal identification of the regulatory T-Cell population. Figure 3a–d: Data shown on CD3⁺ lymphocytes. Figure 3e-f: Data shown on CD3⁺ CD4⁺ lymphocytes.

Maximize the power of your multicolor experiments

Example data from a 7-color TNK panel

Example data from a 7-color TNK panel

Example: Use of CD8 Brilliant Violet 421 in a 7-color panel to identify T cells and NK cells. Lysed whole blood stained with CD8 Brilliant Violet 421, CD3 APC-H7, CD7 PE, CD16 PE-Cy™7, CD45 BD Horizon V500, CD56 APC, and CD57 FITC. This panel was run on a BD FACSVerse flow cytometer.

Example of Brilliant Violet 421 used in an intracellular staining protocol

Example of Brilliant Violet 421 used in an intracellular staining protocol

Peripheral blood mononuclear cells (PBMCs) stimulated with staphylococcus enterotoxin B (SEB) for 6 hours in the presence of brefeldin A (BFA). Cells were then washed, fixed with BD FACS™ lysing solution, permeabilized with BD FACS™ Permeabilizing Solution 2, and stained with CD8 Brilliant Violet 421, CD3 FITC, CD4 PerCP-Cy™5.5, IFN-γ PE, and IL-2 APC. The panel was run on a BD FACSVerse and data is shown on CD3⁺ gated lymphocytes.

Compatible with standard surface and intracellular staining protocols

Brilliant Violet 421 is compatible with standard buffers used in surface and intracellular staining protocols including BD Cytofix™ fixation buffer/BD Perm/Wash™ buffer, Human FoxP3 Buffer Set, BD Phosflow™ Fixation Buffer I/BD Phosflow™ Perm/Wash Buffer I, BD Phosflow™ Perm Buffer II, and BD Phosflow™ Perm Buffer III. The following plots show CD3 Brilliant Violet 421 staining using various staining protocols. Note that staining intensity under various fixation/permeabilization conditions is variable between proteins, so buffer compatibility is a function of the target protein, fluorochrome, and antibody used.

Example of Brilliant Violet 421 staining using various protocols

Example of Brilliant Violet 421 staining using various protocols

Histogram overlays of human CD3 staining using various staining protocols: live-cell stain, cell staining post-fixation/permeabilization, or cell staining prior to fixation/permeabilization.