Products and Services - MACS Flow Cytometry - Reagents - Vio Dye Antibody Conjugates

Vio® Dye Antibody Conjugates

  • Bright: superior mean fluorescence intensity for excellent signals
  • Distinct: high stain index for clear population discrimination
  • Hassle-free: ideal for multicolor experiments due to low compensation

 

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Vio® Dyes represent a family of fluorochromes for flow cytometry and fluorescence microscopy developed by Miltenyi Biotec. VioBlue®, VioGreen™, PE-Vio770™, and APC-Vio770™ Dyes are characterized by high fluorescence intensities and low spillover, making them an ideal choice for multicolor applications. Combined with traditional fluorochromes, such as FITC, PE, PerCP, and APC, the new Vio Dyes expand Miltenyi Biotec’s antibody offering and allow researchers a greater selection of antibodies for multiparameter flow cytometry. 

VioBlue® Dye

Designed to maximize the potential of a flow cytometer’s violet laser, the VioBlue® Dye shows superior performance compared with many other fluorophores excited at 405 nm, including significant advances in brightness, signal-to-noise ratios, and intralaser compensation requirements. In addition, VioBlue exhibits minimal photo-induced degradation, and can consequently be used for many different applications, such as fluorescence microscopy.

 

The VioBlue Dye at a glance:

Table 1: Absorption and emission maxima of fluorochromes related to VioBlue.
The VioBlue Dye at a glance:
  • Coumarin-based dye with excitation and emission wavelengths of 400 nm and 455 nm, respectively
  • Superior alternative to Pacific Blue™, Alexa Fluor® 405, or BD™ Horizon™ V450
  • Multiplexing of VioBlue with other fluorochromes is easily possible, adding to the variety of marker combination for multiparameter flow cytometry
  • Combined use with the VioGreen™ Dye

Laser and filter compatibility

Figure 1: Absorption (top) and emission (bottom) spectra of VioBlue compared to Pacific Blue, Cascade Blue, and Alexa Fluor 405. The blue box represents the 450/50 nm filter.
Laser and filter compatibility
With a maximum absorption and emission at 400 nm and 455 nm, respectively, VioBlue Conjugates are fully compatible with standard filter sets from all major flow cytometry hardware providers, giving researchers the flexibility to use the VioBlue Dye with all existing platforms.

Enhanced brightness

Figure 2: Whole blood was left untreated (left) or stained with CD45-PE (middle) or CD45-VioBlue (right). Plots show data gated on leukocytes. Dead cells were excluded from the analysis.
Enhanced brightness

When compared to well-established fluorochromes with high fluorescence intensities, such as PE, VioBlue exhibits a similar degree of fluorescence (fig. 2).
VioBlue Conjugates provide a superior alternative to many spectrally similar conjugates for the V1 channel, further increasing the options of multicolor analysis (fig. 3).

 

 

 

Figure 3: Fluorescence intensity of cells labeled with CD14 antibodies conjugated to either VioBlue or Pacific Blue.

Decreased spillover

VioBlue Conjugates exhibit minimal spillover into the V2 channel, making them perfect candidates for multicolor panels, which utilize both violet channels. Furthermore, VioBlue is negligibly excited by the 488 nm laser, and thus requires no compensation between the V1 and B1 channels.

High stability during fixation

Figure 4: CD123-VioBlue staining before (left) and after (right) fixation with 3.7% paraformaldehyde indicating only a very small decrease in fluorescence after fixation.
High stability during fixation
It is of crucial importance for a conjugate to retain its fluorescent properties after fixation, in order to allow researchers to maximize the use of biological samples. The VioBlue Dye has a very high stability after fixation with paraformaldehyde (fig. 4), equal to or exceeding many other spectrally similar fluorochromes.

Click here for all VioBlue Conjugates.

Learn more about the Vio Dyes’ technical specifications.

VioGreen™ Dye

Designed to maximize the potential of a flow cytometer’s violet laser, the VioGreen™ Dye shows superior performance compared with many other fluorophores excited at 405 nm, including significant advances in brightness, signal-to-noise ratios, and intralaser compensation requirements. In addition, VioGreen exhibits minimal levels of photo-induced degradation, and can consequently be used for many different applications, such as fluorescence microscopy.

 

The VioGreen Dye at a glance:

Table 1: Absorption and emission maxima of fluorochromes related to VioGreen.
The VioGreen Dye at a glance:
  • Large Stokes shift fluorophore, emitting strong fluorescence at 520 nm upon excitation at 405 nm
  • Significantly increased mean fluorescence intensities and higher stain indices than Pacific Orange™, Krome Orange™, and BD Horizon™ V500
  • Non-protein fluorophore
  • Combined use with the VioBlue® Dye
  • Perfectly suited for multiparameter flow cytometry using all current flow cytometers

Laser and filter compatibility

Figure 1: Absorption (top) and emission (bottom) spectra of VioGreen, Pacific Orange, and AmCyan. The blue box represents the 525/50 nm filter.
Laser and filter compatibility
With a standard 525/50 filter set, VioGreen exhibits a better spectral profile than Pacific Orange or AmCyan.

Enhanced brightness

Figure 2: Whole blood was either left unstained or stained with CD16-PE or CD16-VioGreen. Plots show data gated on leukocytes. Dead cells were excluded from the analysis.
Enhanced brightness

Like most dyes designed for the violet laser, VioGreen shows a lower fluorescence intensity compared to other well-established fluorochromes, such as PE. However, specific cell populations can be distinguished through the identification of unlabeled (fig. 2, left), PE- (middle), or VioGreen-labeled (right) cells.

 

 

Table 2: MFI and stain indices of CD8-VioGreen and CD8-Pacific Orange.

In addition, many VioGreen Conjugates exhibit brighter fluorescence compared to spectrally similar conjugates, including Pacific Orange (fig. 3, table 1), AmCyan, and Horizon V500, measured by mean fluorescence intensity (MFI) or stain index (normalized signal-to-noise ratio).

 

Figure 3: Analysis of human PBMCs using CD8 antibodies (clone BW135/80) conjugated to either VioGreen or Pacific Orange. Concurrent staining with CD14-PerCP and CD56-PE was performed to exclude CD14+ and CD56+ cells from the analysis.

High stability during paraformaldehyde and ethanol fixation

Figure 4: CD14-VioGreen staining before (left) and after (right) paraformaldehyde fixation, indicating only a very small decrease in fluorescence after fixation.
High stability during paraformaldehyde and ethanol fixation
The VioGreen Dye exhibits strong photostability during fixation, with only very minimal photodegradation, thus highlighting VioGreen’s suitability for use in studies that require fixation (fig. 4). It is also suitable for ethanol fixation.

Click here for all VioGreen Conjugates.

Learn more about the Vio Dyes’ technical specifications.

PE-Vio™ Dye

The PE-Vio770™ Dye is a tandem conjugate, like PE-Cy™7, that exploits the principle of fluorescence-resonance-energy-transfer (FRET) allowing for large Stokes shifts between the absorbed energy of a fluorescence donor (PE) and the emission wavelength of a suitable acceptor (Vio770) dye.

 

The PE-Vio770 Dye at a glance

Table 1: Absorption and emission maxima of fluorochromes related to PE-Vio770.
The PE-Vio770 Dye at a glance
  • Excitation with the blue (488 nm) or yellow (561 nm) laser, emission in the near-infrared region at 775 nm.
  • The combination of Vio770 as the acceptor dye and an optimized chemistry have furnished a tandem dye that is characterized by a high fluorescence intensity, minimal spillover to adjacent detection channels, and low non-specific binding to non-target cells to meet the complexity of multiparameter flow cytometry.
  • Higher MFI and stain index values, in addition to lower compensation settings when compared to PE-Cy7.

Laser and filter compatibility

Figure 1: Absorption and emission spectra of PE-Vio770.
Laser and filter compatibility
The tandem dyes PE-Vio770, PE-Cy7, and PE-Alexa Fluor 750 all use PE as the donor molecule, showing absorption at 495 nm and, to a greater extent, at 567 nm. Consequently, the MACSQuant VYB, equipped with a yellow 561 nm laser, is best placed to provide maximum excitation to PE molecules, which in turn transfer this energy to the respective acceptor molecule. Vio770 shows emission properties similar to Cy7, and Alexa Fluor 750.

Enhanced brightness

Figure 2: Whole blood was left untreated (left) or stained with CD45-PE (middle) or CD45-PE-Vio770 (right). Plots show data gated on leukocytes. Dead cells were excluded from the analysis.
Enhanced brightness

The PE-Vio770 Dye provides the greatest fluorescence intensity of the Vio Dye range.
When compared to other spectrally similar tandem conjugates, such as PE-Cy7 (fig. 3) or PE-Alexa Fluor 750, PE-Vio770 exhibits significantly higher mean fluorescence intensities, (MFI) and stain indices, and requires less compensation (table 2). These advantages make PE-Vio770 a far superior tandem conjugate compared to others currently available.

 

Figure 3: Analysis of human PBMCs using CD8 antibodies (clone BW135/80) conjugated to either PE-Vio770 or PE-Cy7. Concurrent staining with CD14-PerCP and CD56-PE was performed to exclude CD14+ and CD56+ cells from the analysis.

 

 

 

 

 

 

 

 

 

 

 

 

Table 2: MFI, stain indices, and compensation requirements of CD8-PE-Vio770 and CD8-PE-Cy7.

High stability during fixation

Figure 4: CD45RA-PE-Vio770 fluorescence before (left) and after (right) paraformaldehyde fixation. MFIs with and without fixation amounted to 124 and 139, respectively, resulting in a decrease in fluorescence of only 11% after fixation.
High stability during fixation
Tandem conjugates are usually less stable after fixation than single-absorption/emission fluorochromes. However, PE-Vio770 shows excellent stability as shown in figure 4.

Click here for all PE-Vio770 Conjugates.

Learn more about the Vio Dyes’ technical specifications.

APC-Vio770™ Dye

The APC-Vio770™ Dye is a tandem conjugate, like APC-Cy™7 or APC-H7, that exploits the principle of fluorescence-resonance-energy transfer (FRET) allowing for large Stokes shifts between the absorbed energy of a fluorescence donor (APC) and the emission wavelength of a suitable acceptor (Vio770).

 

The APC-Vio770 Dye at a glance

Table 1: Absorption and emission maxima of fluorochromes related to APC-Vio770.
The APC-Vio770 Dye at a glance
  • Excitation with the yellow (561 nm) or red (635 nm) laser, emission in the near-infrared region at 775 nm.
  • The combination of Vio770 as the acceptor dye and an optimized chemistry have furnished a tandem dye that is characterized by a high fluorescence intensity, minimal spillover to adjacent detection channels, and low non-specific binding to non-target cells to meet the complexity of multiparameter flow cytometry.
  • Higher MFI and stain index values, in addition to lower compensation settings when compared to APC-Cy7.

Laser and filter compatibility

Figure 1: Absorption and emission spectra of APC-Vio770.
Laser and filter compatibility
The tandem dyes APC-Vio770, APC-Cy7, and APC-H7 all use APC as the donor fluorochrome, showing maximum absorption around 652 nm. Emission spectra for Vio770, Cy7, H7, and Alexa Fluor 750 are similar. Therefore, APC-Vio770 is an ideal tandem conjugate candidate for this channel in all flow cytometers.

Enhanced brightness

Figure 2: The strong staining pattern of APC-Vio770 can be illustrated by comparing unstained (left), CD16-APC-stained (middle), and CD16-APC-Vio770-stained (right) cellular material, after gating on leukocytes and dead cell exclusion.
Enhanced brightness

APC-Vio770 provides strong staining, allowing the identification and analysis of specific cell populations (fig. 2).
When compared to other spectrally similar conjugates, such as APC-Cy7 and APC-H7, APC-Vio770 exhibits equal or stronger staining patterns (fig. 3). In addition, APC-Vio770 generally exhibits higher mean fluorescence intensities (MFI) and greater stain index values, and requires less compensation in the R1 channel than both APC-Cy7 and APC-H7 (table 2). These properties make APC-Vio770 an ideal fluorochrome for use in the R2 channel.

 

Figure 3: Analysis of human PBMCs using CD8 antibodies (clone BW135/80) conjugated to APC-Vio770, APC-Cy7, or APC-H7. Concurrent staining with CD14-PerCP and CD56-PE was performed to exclude CD14+ and CD56+ cells from the analysis.

 

 

 

 

 

 

 

 

 

 

 

 

Table 2: MFI, stain indices, and compensation requirements of CD8-APC-Vio770, CD8-APC-Cy7, and CD8-APC-H7.

High stability during fixation

Figure 4: Cells were stained with CD45-APC-Vio770 and left untreated (left) or fixed with paraformaldehyde (right). MFIs with and without fixation amounted to 62 and 68, respectively, resulting in a decrease in fluorescence of only 8% after fixation.
High stability during fixation
APC-Vio770 shows excellent stability after fixation with paraformaldehyde (fig. 4), similar to PE-Vio770.

Click here for all APC-Vio770 Conjugates.

Learn more about the Vio Dyes’ technical specifications.

PerCP-Vio700 Dye

The PerCP-Vio700 Dye is a tandem conjugate that combines the peridinin chlorophyll protein (PerCP) and the new Vio700 Dye to emit a strong fluorescence at 655–730 nm upon blue laser excitation at 488 nm.  This dye is suited perfectly for the B3 channel of the MACSQuant® Analyzer.

The PerCP-Vio700 Dye at a glance

Table 1: Absorption and emission maxima of fluorochromes related to PerCP-Vio700.
The PerCP-Vio700 Dye at a glance

Laser and filter compatibility

Figure 1: Absorption and emission spectra of PerCP-Vio700. The blue box represents the 655–730 filter.
Laser and filter compatibility
Using a standard 655–730 bandpass filter, PerCP-Vio700 exhibits a very narrow fluorescence spectral profile, thus allowing the majority of light to be captured and retained (fig. 1).

Enhanced brightness

Figure 2: Human peripheral blood mononuclear cells (PBMC) or mouse splenocyte (MS) cells were stained with CD20-PerCP-Vio700 (A; PBMCs), CD4-PerCP-Vio700 (B; PBMCs), CD45-PerCP-Vio700 (C; PBMCs) or CD90.2-PerCP-Vio700 (D; MS) and analyzed by flow cytometry using the MACSQuant Analyzer.
Enhanced brightness
Human peripheral blood mononuclear cells (PBMC) and mouse splenocyte (MS) cells were stained with PerCPVio700, and consequently exhibited excellent separation between positive and negative populations over many different antibody specificities (fig. 2).

High stability during fixation

Figure 3: CD8-PerCP-Vio700 before (A) and after (B) fixation with 3.7% paraformaldehyde, indicating a minimal decrease in fluorescence after fixation.
High stability during fixation
PerCP-Vio700 shows excellent fixation stabilities, with only minimal decreases in fluorescence after fixation (fig. 3).

Photo-induced conjugate degradation

Figure 4: Photo-induced conjugate degradation of PerCP, PerCP‑Vio700, and PerCP-Cy5.5 with corresponding MFI and SI. Conjugates were exposed to ambient light for up to four hours, with negligible degradation rates.
Photo-induced conjugate degradation
Analysis of the photo-induced degradation of CD14-PerCPVio700 indicated no discernable changes after up to four hours of continuous exposure to ambient light (~850 Lux). Significantly higher mean fluorescence intensities (MFI) and stain indices (SI) for these time points, compare to commercially available alternatives, were also demonstrated (fig. 4).

Click here for all PerCP-Vio700 Conjugates.

Learn more about the Vio Dyes’ technical specifications.

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