BD Horizon Brilliant™ Blue Dyes

More Brilliance for the Blue Laser

Optimally resolve dim populations with brighter options for the blue laser. BD Horizon Brilliant™ Blue 515 (BB515) is brighter than FITC, and BD Horizon Brilliant™ Blue 700 (BB700) is a brighter alternative to PerCP-Cy™5.5.

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BB700 Spectra

Figure 1. Absorption and emission spectra of BB700—Ex Max: 485 nm, Em Max: 693 nm

A brighter alternative to PerCP-Cy5.5

BB700 was developed as a brighter alternative to PerCP-Cy5.5, making it better suited for resolving dim populations. Figure 1 shows the absorption and emission spectra of BB700. Figure 2c shows how a dimmer dye such as PerCP-Cy5.5 could underestimate the CD279 expression, while a bright dye such as BB700 is able to fully resolve the CD279 + cells, leading to more accurate results. Having an additional bright dye for the blue laser expands the choices available for resolving dim populations.

BB700 - Fig2 Hu CD4 Comparison

Figure 2. (A) Lysed whole blood stained with Hu CD4 BB700 (clone SK3, BD Biosciences, blue), PerCP-Cy5.5 (clone SK3, BioLegend, green), PerCP-eFluor® 710 (clone SK3, eBioscience, red) or PerCP-Vio®700 (clone REA623, Miltenyi, brown) using the manufacturer’s recommended volume per test. (B) Lysed whole blood stained with Hu CD19 BB700 (clone SJ25C1, BD Biosciences, blue), PerCP-Cy5.5 (clone SJ25C1, BioLegend, green), PerCP-eFluor® 710 (clone SJ25C1, eBioscience, red) or PerCP-Vio®700 (clone REA675, Miltenyi, brown) using the manufacturer’s recommended volume per test. (C) Peripheral blood mononuclear cells (PBMCs) were stained with CD279 PerCP-Cy5.5 or BB700 and CD8 BV510. Data shown is gated on CD3 + cells.

BB700 - Hu PBMC CD3

Figure 3. Human PBMCs were stimulated with anti-CD3 and anti-CD28 for 72 hours and stained with PE Mouse Anti-Human LAG-3 and either BB700 (left plot) or PerCP-Cy5.5 (right plot) Mouse Anti-Human PD-1. The cells were also stained with BD Via-Probe™ red nucleic acid stain for live/dead cell discrimination. The two-color dot plots showing correlated expression of PD-1 vs LAG-3 were derived from gated-events characteristics of BD Via-Probe red live cells.

Spillover advantages to optimize panel design

BB700 has less cross-laser excitation on the 405-nm and 561-nm lasers compared to PerCP-Cy5.5, resulting in less spillover into multiple channels, making BB700 more useful for multicolor panels (Table 1). With an excitation max at 485 nm and emission max at 693 nm, BB700 can be excited by the blue laser (488 nm) and detected in the same filter as PerCP-Cy5.5 (for example, 695/40 nm).

BB700 - Spillover Comp Table

Table 1. Human CD4 reagents conjugated to various fluorochromes run side by side for a spillover comparison
All data was collected on a BD LSR Fortessa™ X-20 system. To collect data across the most channels, the data from the UV, violet and blue lasers came from one instrument and the data from the red and yellow-green lasers came from another instrument. This table is meant to show a relative comparison between the dyes, since spillover values obtained can vary depending on the filter used and photomultiplier tube (PMT) voltage.

Compatible with standard surface and intracellular staining protocols

BD Horizon BB700 is compatible with standard buffers used in surface and intracellular staining protocols. When cells are stained prior to the permeabilization step in the presence of strong alcohol-based buffers such as BD Phosflow™ perm buffer III, PerCP-Cy5.5 staining is no longer detectable. However, BB700 is compatible with BD Phosflow perm buffer III, making it an ideal choice for intracellular staining conditions (Figure 4).

BB700 - Perm Buffer

Figure 4. Lysed whole blood was stained with Hu CD4 BB700 (clone SK3, BD Biosciences, blue), PerCP-Cy5.5 (clone SK3, BD Biosciences, green), PerCP-eFluor® 710 (clone SK3, eBioscience, red) or PerCP-Vio®700 (clone REA623, Miltenyi, brown) using the manufacturer’s recommended volume per test, washed, incubated with BD Phosflow perm buffer III, washed and run on a cytometer (right). The only reagent that shows staining is CD4 BB700, due to its compatibility with the permeabilization buffer. As a control, lysed whole blood was stained with the same reagents but not incubated with BD Phosflow perm buffer III (left).

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For Research Use Only. Not for use in diagnostic or therapeutic procedures.
BD flow cytometers are Class 1 laser products.

BB515 Spectra

Figure 1. Absorption and emission spectra—Ex Max: 490, Em Max: 515

A brighter alternative to FITC

BD Horizon™ BB515 is a brighter alternative to FITC, making it more optimal for dimmer markers, such as CD25, for which better resolution improves the quality of a panel. Figure 1 shows the absorption and emission spectra of BB515. CD25 FITC or CD25 BB515 was used to identify regulatory T cells (Tregs) in a panel including CD4 APC, CD127 PE and CD3 PerCP-Cy5.5. While both panels resolve the Treg population, the panel including CD25 BB515 shows significantly better separation of the CD25 + cells from the CD25 cells (Figure 2).

BB515 - CD3 CD19 FITC

Figure 2. Lysed whole blood stained with Hu CD4 APC, CD127 PE, CD3 PerCP-Cy5.5 and CD25 FITC or CD25 BB515
Data shown was gated on CD4 +CD3 + lymphocytes.

BB515 - CD4 APC

Figure 3. Lysed whole blood stained with Hu CD3 or CD19 FITC (red), BB515 (blue) or Alexa Fluor® 488 (green)
Data shown was gated on lymphocytes.


Increased brightness for better resolution

 
Stain Index
  BB515 FITC Alexa Fluor® 488
Hu CD3 302 43 81
Hu CD4 174 47 58
Hu CD19 85 16 15
MS CD8a 86 24 50
MS CD11b 68 15 26

Table 1. BD Horizon BB515, Alexa Fluor® 488 and FITC reagents of the same clone run side by side to compare the stain index

BB515 has less spillover into neighboring channels, providing more choices for optimal panel design

 
Spillover Into
  BV510 PE PE-CF594
Hu CD4 BB515 2% 20% 6%
Hu CD4 FITC 6% 27% 9%

Table 2. Spillover into various detectors, a comparison of BD Horizon BB515 and FITC
Whole blood samples stained with human CD4 BB515 or FITC were analyzed on a BD LSRFortessa™ system, and spillover was measured in the BV510, PE and PE-CF594 detectors. This table is meant to show a relative comparison between the dyes, since spillover values obtained can vary depending on the filter used and PMT voltage.

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Alexa Fluor® is a registered trademark of Life Technologies Corporation. Cy™ is a trademark of GE Healthcare.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
BD flow cytometers are Class 1 laser products.

Use of BB515 and BB700 in a multicolor panel for the analysis of immune checkpoint expression

T-cell activation is tightly regulated by immune checkpoints, a combination of co-stimulatory and co-inhibitory signals capable of promoting or suppressing T-cell response, respectively. An eight-color panel was designed to assess the expression of co-stimulatory receptors CD137 (4-1BB) and CD134 (OX40), as well as co-inhibitory receptors CD279 (PD-1) and CD366 (TIM- 3), within the CD4 + and CD8 + subsets of phytohemagglutinin (PHA)-stimulated CD3 + T cells. The expression of co-signaling receptors is modulated upon T-cell activation, and the level of expression within different subset of cells can be variable. Similarly, the expression of CD3 is known to be significantly down-modulated upon T-cell stimulation. All these factors were taken into consideration when designing the panel. The use of bright dyes (BV421, BB700, APC and PE) allowed for optimal resolution of all the co-signaling receptors in both CD4 and CD8 subsets. As shown in Figure 5c, use of a dim dye such as PerCP-Cy5.5 would underestimate the percentage of cells expressing CD279, so BB700 was chosen instead. In most cases, FITC is an optimal choice for a highly expressed marker such as CD3.

However, knowing that CD3 will be down-modulated upon PHA stimulation, BB515 was chosen to provide optimal resolution. As a result, up-regulation of all co-stimulatory and co-inhibitory receptors was detected in PHA-stimulated T cells, compared to untreated control (not shown). The multiparameter approach also allowed for the detection of differences in the level of expression of co-signaling receptors within CD4 + and CD8 + subsets. For example, the co-stimulatory receptor CD134 was highly expressed on CD4 + cells, but dimly on CD8 + (Figure 1).

Sample Data - Co-signaling Receptor Expression

Figure 1. Multicolor panel for the analysis of co-signaling receptor expression
PBMCs were isolated from healthy donors and stimulated with 2% PHA (Sigma) for 12 hours. At the end of the stimulation, cells were collected and stained with a cocktail of antibodies, including CD3 BB515 (Cat. No. 564465), CD4 BV786 (Cat. No. 563877), CD8 BV510 (Cat. No. 563256), CD279 BB700 (Cat. No. 566460), CD366 BV421 (Cat. No. 565562), CD134 PE (Cat. No. 555838) and CD137 APC (Cat. No. 550890), in the presence of BD Horizon™ Brilliant Stain Buffer (Cat. No. 563794). Cells were then stained with FVS575V viability dye (Cat. No. 565694). Cells were analyzed on a BD FACSCelesta™ system with a Blue/Violet/Red configuration. CD3 + T cells were further divided into CD4 + and CD8 + subsets. Each subset was interrogated for the expression of co-stimulatory and co-inhibitory receptors. While the expression of all the signaling receptors was increased overall as compared to unstimulated cells (not shown), a difference in the frequency and level of expression within CD4 + and CD8 + subsets was observed.

Panel-design considerations: save bright fluorochromes for the dimmer markers of the panel

Although BB700 PerCP-Cy5.5 and BB515 FITC are detected in the same channel, the differences in brightness should be taken into account when incorporating the fluorochromes into panels. PerCP-Cy5.5 and FITC should be used for high- or medium-expressed markers in your panel. However, when incorporating BB700 or BB515 into your panel, match the dyes with the dimmer markers in the panel to get the most benefit. Additionally, the differences in brightness should be taken into account when maximizing population resolution. The resolution for a given antigen (fluorescence parameter) is decreased by the spread due to spillover from other fluorochromes. That is, the addition of a reagent may reduce the resolution of another reagent, potentially affecting overall population resolution and data quality. Spread is most important when considering reagents for co-expressed antigens. It is a not only a function of spillover but also antigen density and fluorochrome brightness. Therefore, on the same antigen, a fluorochrome that has less spillover but is also significantly brighter may actually cause more spread. For example, if comparing BB700 and PerCP-Cy5.5 on markers of equal antigen density, the spread may be similar or wider with BB700, despite it having less spillover (Figure 2). Figure 2b shows that if the BB700 were titrated down to the brightness of PerCP-Cy5.5 (to mimic switching to a lowly expressed antigen), the reduced spread can be observed. This is also a consideration with BB515, which has less spillover into the PE channel but is significantly brighter than FITC. If using these fluorochromes on highly expressed antigens, keep in mind the potential spread that may be observed due to their brightness. We recommend saving these fluorochromes for the lower-expressed antigens of the panel where the brightness will help fully resolve the population and is not likely to affect the resolution of other reagents.

Sample Data - BV711 and PE-Cy7

Figure 2. (A) The spread into the BV711 and PE-Cy™7 channels from Hu CD4 BB700 (blue) and PerCP-Cy5.5 (red) is shown using the optimal concentration of each reagent. The spread can be seen as the width of the BB700 PerCP-Cy5.5 + populations. (B) Hu CD4 BB700 (blue) was titrated down to a median fluorescence intensity (MFI) similar to the PerCP-Cy5.5 reagent (red). At similar MFI target values, the spread of BB700 into BV711 and PE-Cy7 is similar to or less than that of PerCP-Cy5.5. PE-Cy7 was detected off the 561-nm laser.


Cy™ is a trademark of GE Healthcare.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
BD flow cytometers are Class 1 laser products.


For Research Use Only. Not for use in diagnostic or therapeutic procedures.

Cy™ is a trademark of GE Healthcare.

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