• High purity and lot-to-lot consistency for greater reproducibility
  • Eliminates tedious and costly Fc receptor-blocking steps
  • One universal isotype control for convenience and cost savings

In a recent commentary in Nature, Andrew Bradbury and Andreas Plückthun highlighted the need for recombinant antibodies to improve reproducibility in research. This way, the use of recombinant antibodies will also help save research funding. REAfinity™ Antibodies are recombinant antibodies that provide superior lot-to-lot consistency and purity compared to mouse or rat monoclonal antibodies. They have been recombinantly engineered for high specificity and require no FcR blocking step. Additionally, they all have the same IgG1 isotype. 

REAfinity™ Antibodies offer several major advantages over mouse monoclonal antibodies for long-term studies, including:

  1. REAfinity Antibodies contain only one heavy and one light chain for higher lot-to-lot consistency.
  2. The unmatched quality of REAfinity Antibodies also arises from its manufacture in mammalian cells and production under a highly controlled manufacturing process.
  3. Compared to the average mouse monoclonal antibody, REAfinity Antibodies offer greater purity and higher lot-to-lot consistency to make REAfinity Antibodies your reagent of choice.
old verses new way to make antibodies

What are the differences between traditional monoclonal and recombinant monoclonal antibodies?

To generate traditional monoclonal antibodies, a host animal is immunized with an immunogen and the antibody-producing B cells are isolated. An immortalized antibody-producing hybridoma cell line is then generated by fusing the B cells with myeloma cells. Several clones of hybridoma fusion cells are screened to select a monospecific antibody-producing clone. Once established, this fusion cell line clone is used for the production of monoclonal antibodies over a long period of time. Long-term use however makes the hybridomas susceptible to genetic drift potentially leading to a change in the antibody sequence and hence affecting the performance of the antibody. In addition, antibodies derived from hybridomas are contaminated with i) immunoglobulin impurities derived from serum used in the culture media and ii) immunoglobulin light chains produced by myeloma cells. 

Recombinant antibodies on the other hand are produced by cells that normally do not express antibodies by themselves. Antibody production in these cells is only driven by the expression vector containing the desired antibody genes. Therefore, these cells produce highly pure antibodies. In addition, standardized culture conditions ensure lack of contaminating immunoglobulins. Since the antibody sequence is exactly defined, the antibodies produced are also highly defined in nature and any shift in performance can be traced back by sequencing the antibodies. Due to their excellent purity, the standardized culture conditions, and the absence of animal-derived components during cell culture, recombinant antibodies are highly consistent in their structure and performance.


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Recombinant antibodies for improved reproducibility in flow cytometry analysis


What are recombinant antibodies?

Recombinant antibodies are derived from a defined genetic sequence and generated in vitro. Sources of the antibody-encoding gene are either antigen-stimulated B cells or hybridomas, the latter of which are an excellent source of monospecific antibodies. Alternatively, the gene sequence is generated synthetically. The antibody-specific gene is then cloned into a high-expression vector. Subsequently, the antibody is produced in a suitable expression system such as mammalian cells.
REAfinity™ Antibodies from Miltenyi Biotec, for example, are recombinantly engineered antibodies. The variable region of the REAfinity Antibodies is derived from hybridoma clones established to produce highly specific antibodies, and the constant region is human IgG1. 

What are the differences between traditional monoclonal and recombinant monoclonal antibodies?

To generate traditional monoclonal antibodies, a host animal is immunized with an immunogen and the antibody-producing B cells are isolated. An immortalized antibody-producing hybridoma cell line is then generated by fusing the B cells with myeloma cells. Several clones of hybridoma fusion cells are screened to select a monospecific antibody-producing clone. Once established, this fusion cell line clone is used for the production of monoclonal antibodies over a long period of time. Long-term use however makes the hybridomas susceptible to genetic drift potentially leading to a change in the antibody sequence and hence affecting the performance of the antibody. In addition, antibodies derived from hybridomas are contaminated with i) immunoglobulin impurities derived from serum used in the culture media and ii) immunoglobulin light chains produced by myeloma cells. 
Recombinant antibodies on the other hand are produced by cells that normally do not express antibodies by themselves. Antibody production in these cells is only driven by the expression vector containing the desired antibody genes. Therefore, these cells produce highly pure antibodies. In addition, standardized culture conditions ensure lack of contaminating immunoglobulins. Since the antibody sequence is exactly defined, the antibodies produced are also highly defined in nature and any shift in performance can be traced back by sequencing the antibodies. Due to their excellent purity, the standardized culture conditions, and the absence of animal-derived components during cell culture, recombinant antibodies are highly consistent in their structure and performance.

High consistency and reproducibility

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Recombinant antibodies are derived from a defined set of genes, and the antibody production process is highly standardized. Therefore, these antibodies are consistent in their structure and performance, leading to high experimental reproducibility and lot-to-lot consistency (fig. 1).

High purity – no IgG chain mixtures

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Recombinant antibodies generated in artificial expression systems do not contain contaminants from serum and myeloma cell–derived immunoglobulin light chains. Therefore, these antibodies are highly pure. Contaminating Ig chains which are mixtures of different IgG heavy and light chains (fig. 1), are commonly present in traditional antibody preparations. This impurity can not only result in lot-to-lot variation but also affect the specificity of the antibody and could impact the reproducibility of your experiments.

Eliminate unwanted Fcγ receptor binding

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Since the gene sequence of a recombinant antibody is known, it is easy to modify the sequence and improve the properties of the antibodies. For example, REAfinity™ Antibodies have specifically mutated human IgG1 Fc regions. These mutations virtually eliminate non-specific binding to Fcγ receptors, as demonstrated in figure 1. Thus, REAfinity Antibodies allow for a higher signal specificity during cell analysis and make the use of FcR blocking reagents redundant resulting in time and cost savings.

In the absence of an FcR blocking reagent, staining with the mouse monoclonal antibody results in strong non-specific background signals (figure 5A). Therefore, it is necessary to include an FcR blocking step prior to staining with the mouse monoclonal antibody (figure 5B). In contrast, staining with the REAfinity Antibody allows the specific detection of the CD158a+ target population, even without FcR blocking reagent (figure 5C).

One universal isotype control

REAfinity™ Antibodies all have the human IgG1 isotype in common. The result is that only one type of isotype control is required for all REA clones, named REA Control Antibodies or clone REA293. This control is available in two formats, one for surface antigens and one for intracellularly expressed antigens.

What does one isotype control per fluorochrome really mean?

  • Significant cost savings, since the use of only one control is sufficient.
  • Time usually spent on searching for the antibody isotype can be spent on other tasks

See performance data

We back up our claims that REAfinity™ Antibodies are the best antibodies available with real data. They are demonstrated to be highly specific, result in better staining than mouse or rat monoclonals, and without the need of Fcγ receptor blocking step.

  • Highly specific: better staining than mouse and rat monoclonals
  • Clear resolution of target population: enables straightforward data analysis
  • High stain index: due to an optimized fluorochrome labeling process and low background

Figure 1: Specific detection of CD158b2+ cells with REA147. PBMCs were stained with a PE-conjugated REAfinity Antibody (A) and mouse monoclonal PE-conjugated antibody (B) recognizing CD158b2. Additionally, staining with CD56-APC (# 130-100-698) was performed followed by flow cytometry analysis on the MACSQuant® Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide (PI) fluorescence.

Further reading

1. Bradbury, A. and Plückthun, A. (2015) Reproducibility: Standardize antibodies used in research. Nature. 518: 27–29.
2. Geyer, C.R. et al. (2012) Recombinant antibodies and in vitro selection technologies. Methods Mol Biol.  901: 11–32.
3. Frenzel, A. et al. (2013) Expression of recombinant antibodies. Front Immunol. 4: 217.
  • 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
Figure 1: Emission spectra of VioDyes: VioBlue, VioBright 515, Vio515, VioGreen, VioBright FITC, PE-Vio615, PerCP-Vio700, PE-Vio770, APC-Vio770
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Vio® Dyes technical specifications

Violet Laser (405) nm - 
VioBlue® Dye (Emmax 452 nm)

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:

  • 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
Table 1: Absorption and emission maxima of fluorochromes related to VioBlue.
FluorochromesAbsorpotion max. (nm)Emission max. (nm)

VioBlue

400455
Pacific Blue405455
Cascade Blue(375); 401423
Alexa Fluor 405405421
eFlour 405405450
BD Horizon V450404448
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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.

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Enhanced brightnes

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).

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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.

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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.

Violet Laser (405 nm) -
VioGreen™ Dye (Emmax 520 nm) 

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:

  • 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
Table 1: Absorption and emission maxima of fluorochromes related to VioGreen.
FluorochromeAbsorption max. (nm)Emission max. (nm)
VioGreen388520
Pacific Orange400551
Krome Orange398528
AmCyan458489
Horizon V500415500
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Laser and filter compatibility

With a standard 525/50 filter set, VioGreen exhibits a better spectral profile than Pacific Orange or AmCyan.

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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. 

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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).

Table 2: MFI and stain indices of CD8-VioGreen and CD8-Pacific Orange.
SampleConjugateMFIStain index
ACD8-VioGreen12.112.7
ACD8-Pacific Orange6.48.4
BCD8-VioGreen11.311.9
BCD8-PacificOrange6.17.0
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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.

Blue Laser (488nm) -
VioBright™ 515 (Emmax 514 nm)

VioBright™ 515 is an extremely bright dye for the B1/FITC-channel. The proprietary VioBright multimerization technology results in a 4-fold increase in brightness over the traditional FITC dye.

VioBright™ 515 at a glance:

  • Brightest VioDye for the blue laser (488 nm)
  • Excellent choice for surface markers, in particular for low expressed antigens
  • Spill-over into B2/PE-channel reduced by 25%
  • High photo-stability for immunofluorescence microscopy applications
Table 1: Absorption and emission peaks of fluorochromes compared to VioBright™ 515.
FluorochromAbsorption max. (nm)Emission max. (nm)
VioBright 515488514
Vio 515488514
VioBright FITC496522
FITC495520
Alexa Fluor 488495519
BD Horizon Brilliant Blue 515490515
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Laser and filter compatibility

Upon blue laser excitation (488 nm), VioBright™ 515 displays peak excitation and emission at 488 nm and 514 nm, respectively. Its fluorescent signal can be detected with a standard FITC filter, such as the 525/50 of the MACSQuant® instruments. Thus, no changes in detection filters or the cytometer itself are required. With VioBright 515 the most commonly used laser-filter combination in flow cytometers can be extended to detect a wider variety of markers, including intracellular markers.

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Enhanced brightness

VioBright™ 515 is the best choice for the detection of lowly expressed markers, as shown in figures 2 and 3. With stain indices (SI) and mean fluorescent intensities (MFI) higher than PE, VioBright 515 staining allows for an excellent resolution of positive and negative populations.

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enhanced brightness second data plot ........

Table 2: Mean fluorescent intensities (MFI) and stain indices of anti-CD56 antibodies conjugated to FITC, Viobright™ FITC, VioBright 515, and BB515
ConjugateCloneMFIStain Index

MACSQuant Compensation

in Channel B2 (%)

CD56-FITCREA19610.618.36.6
CD56-VioBright FITCAF1215.822.68.6
CD56-VioBright 515REA19631.956.34.2
CD56-BB515B1594.94.46.2
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Higher specificity – better resolution

VioBright™ 515 is an optimal tool for the analysis of dim markers, such as CD56. In this example from whole blood the CD56+CD3+ NKT cell population could be separated more precisely (right dot-plot) as opposed to an alternative bright dye for the same channel (left dot plot).

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Fixation stability

Bright fluorochrome conjugates, such as PE and APC, are often sensitive to fixatives like Methanol. For a successful flow cytomeric analysis, which often involves staining of intracellular markers, stability of the fluorochrome conjugates is critical. VioBright 515 conjugates show excellent stability to methanol- and paraformaldehyde-based fixatives with only little compromise in brightness (table 3).

Table 3: Human PBMCs were stained using CD56 antibodies (clone REA196) conjugated to VioBright™ 515 and PE. In addition, cells were analyzed before and after fixation using paraformaldehyde and 90% methanol. Stained cells were analyzed by flow cytometry using the MACSQuant® 10.
No FixativePFA FixationMethanol Fixation
ConjugateCloneMFIStain indexMFIStain indexMFIStain index
CD56-VioBright 515REA19623.937.9711.2729.9921.7733.23
CD56-PEREA19611.2723.133.397.002.695.33
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Photostability

The stability of VioBright 515 upon exposure to confocal laser light was analyzed at different time points on a Zeiss LSM710 confocal microscope. Significantly higher mean fluorescence intensity (MFI) compared to FITC conjugate, were demonstrated for these time points (figure 6). VioBright 515 shows comparable stability to Alexa Fluor 488 conjugated antibodies indicating the excellent suitability for immunofluorescence microscopy.

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second dot plot for above text ......

Blue Laser -
Vio®515 (Emmax 514 nm)

Vio® 515 is an organic small molecule dye for the detection of intracellular markers in the B1/FITC-channel.

Vio 515 at a glance

  • Brighter alternative to FITC for intracellular markers, such as cytokines and transcription factors
  • Spillover into B2/PE-channel reduced by 25%
  • High photo-stability for immunofluorescence microscopy applications
Table 1: Absorption and emission peaks of fluorochromes compared to Vio® 515.
FluorochromAbsorption max. (nm)Emission max. (nm)
VioBright 515488514
Vio 515488514
VioBright FITC496522
FITC495520
Alexa Fluor 488495519
BD Horizon Brilliant Blue 515490515
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Laser and filter compatibility

Upon blue laser excitation (488 nm), Vio® 515 displays peak excitation and emission at 488 nm and 514 nm, respectively. Its fluorescent signal can be detected with a standard FITC filter, such as the 525/50 of the MACSQuant® instruments. Thus, no changes in detection filters or the cytometer itself are required. With Vio 515 the most commonly used laser-filter combination in flow cytometers can be extended to detect a wider variety of markers, including intracellular markers.

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Enhanced brightness

With stain indices (SI) and mean fluorescent intensities (MFI) higher than FITC, Vio® 515 staining allows for an improved resolution of positive and negative populations as shown in figure 2.

ConjugateCloneMFIStain index

MACSQuant Compensation

in Channel B2 (%)

CD4-FITCVIT425.350.06.4
CD4-Alexa488VIT435.768.25.6
CD4-Vio515VIT435.971.24.4
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Higher specificity – better resolution

Vio® 515 is an optimal reagent for the analysis of intracellular markers, i.e. IL-5 in cytokine-expressing T cells. In this example from PFA-fixed human PBMCs, an Anti-IL-5-Vio515 antibody enabled the detection of an IL-5-expressing CD4+CD69+ T cell subset.

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Photostability

The stability of Vio® 515 upon exposure to confocal laser light was analyzed at different time points on a Zeiss LSM710 confocal microscope. Vio 515 shows a comparable stability to Alexa Fluor 488 conjugated antibodies indicating an excellent suitability for immunofluorescence microscopy (figure 4). A significantly higher mean fluorescence intensity (MFI) compared to FITC conjugate antibodies, was demonstrated at different time points (figure 5).

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second dot plot for above text ......

Blue Laser (488nm) -
VioBright™ FITC (Emmax 522 nm)

VioBright™ FITC

VioBright™ FITC is a blue laser (488 nm) excited revolutionary dye, which allows an increased number of FITC molecules per antibody, as compared to conventional FITC conjugation. With brightness similar to PE, VioBright FITC expands the dimensions of multicolor flow analysis. In addition, it provides a bright alternative for confident detection of rare cells, as well as dim and uncharacterized markers.

VioBright FITC at a glance:

  • Brightness similar to PE for confident detection of low-expressed and rare markers
  • Excellent bright alternative to PE for flexible multi color panel design
  • Signal detectable in the standard FITC channel with peak excitation and emission wavelength of 496 nm and 522 nm, respectively
  • Works with your standard staining protocol
Table 1: Absorption and emission maximums of fluorochromes comparable to VioBright FITC
FluorochromeAbsorption max. (nm)Emission max. (nm)
VioBright FITC496522
FITC495520
Alexa Fluor 488495520
BD Horizon Brilliant Blue 515490515
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Laser and filter compatibility

Upon blue laser excitation (488 nm), VioBright FITC displays peak emission and excitation at 496 nm and 522 nm, respectively. Its high-intensity fluorescent signal can be detected in a standard FITC filter, such as 525/50 of MACSQuant Instruments. Thus, no change in the detection filter or cytometer is required. Along with PE, VioBright FITC enhances the potential of the blue laser, one of the most common laser lines available for single- to multi-laser instruments.

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Enhanced brightness

VioBright FITC is a superior choice of conjugate for detection of low-expressed markers, as shown in figure 2 and 3. With stain indices (SI) and mean fluorescent intensities similar to PE, VioBright FITC staining allows for an excellent resolution of positive and negative population.

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second dot plot for above text ......

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Reliable analysis of rare cells requires specific identification of low frequency cellular subset and clear target population resolution. Thus, highly specific antibodies together with bright conjugates are a prerequisite for detection of such low frequency populations. As demonstrated in figure 4, VioBright FITC offers you the bright dye alternative for optimal detection of your subpopulation of interest and detailed phenotypic analysis.

Table 2: MFI and stain indices of CD25 (clone 4E3), CD335 (clone 9E2), CD133/1 (clone AC133) antibodies conjugated to either VioBright FITC or PE.

Conjugate

MFIStain Index
VioBright FITCPEVioBright FITCPE
 CD253.32.05.04.0
CD3354.84.08.511
CD133/13.54.74.74.7

Spill over

Compared to standard FITC, VioBright FITC displays only a minor increase in spill over into the PE channel. Thus, with nominal change in compensation settings, VioBright FITC provides the benefit of a bright dye.

Table 3: MFI and stain indices of CD4 (clone VIT4) antibodies conjugated to VioBright FITC, FITC, or PE. The staining was done with PBMCs from two independent donors (Sample A and Sample B).
SampleConjugateMFIStain indexMACSQuant Analyzer 10MACSQuant VYB

Compensation in

channel B2 (%)

Compensation in

channel Y1 (%)

ACD4-VioBright FITC661068.50
ACD4-FITC29496.50
ACD4-PE58126--
BCD4-VioBright FITC52819.30
BCD4-FITC20357.00
BCD4-PE3973--

Blue Laser (488nm) -
PerCP-Vio®700 (Emmax 704 nm)

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
FluorochromeAbsorption max. (nm)Emission max. (nm)
PerCP482675
PerCP-Vio700482704
PerCP-Cy5.5490695
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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).

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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).

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High stability during fixation

PerCP-Vio700 shows excellent fixation stabilities, with only minimal decreases in fluorescence after fixation (fig. 3).

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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).

MarkerCloneStain index
t 0 mint 120 mint 240 min
CD14-PerCPTÜK4534436
CD14-PerCP-Vio700TÜK4757575
CD14-PerCP-Cy5.561D3363128

Blue Laser (488nm)/Yellow Laser (561 nm) -
PE-Vio®770 (Emmax 770 nm)

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

  • 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.

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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.

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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.

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second plot for above text


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

SampleConjugateMFIStain index

Compensation in

channel B2 (%)

ACD8-PE-Vio770109.297.30.4
ACD8-PE-Cy767.072.72.2
BCD8-PE-Vio770101.0112.00.4
BCD8-PE-Cy765.681.72.2
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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.

Blue Laser (488nm)/Yellow Laser (561 nm)  -
PE-Vio®615 (Emmax 619 nm)

PE-Vio® 615 is a tandem dye with PE as the donor dye and Vio® 615 as the acceptor dye. This tandem dye is optimized for efficient donor-to-acceptor dye energy transfer, high fluorescent intensity, and low spillover into the donor dye detection channel. Designed to be a superior alternative to ECD, PE-Texas Red®, PE-efluor® 610, PE-CF594 and PE/Dazzle™ 594, this Vio Dye expands the options for flexible multicolor panel design and provides a bright dye for confident detection of dim and rare markers.

PE-Vio615 at a glance:

  • Optimal excitation with blue (488 nm), green (532 nm), and yellow green (561 nm) laser lines for maximum flexibility. 
  • Excellent brightness for confident detection of dim, rare, and uncharacterized markers.
  • Extensive portfolio of PE-Vio615 dye conjugated to recombinantly engineered REAfinity Antibodies for higher reproducibility.
Table 1: Absorption and emission maximums of fluorochromes comparable to PE-Vio615
FluorochromeAbsorption max. (nm)Emission max. (nm)
PE-Vio615565619
ECD, PE-Texas Red565613
PE-efluor 610565606
PE-CF594565614
PE/Dazzle 594566612
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Laser and filter compatibility
The PE molecule shows peak absorption at two wavelengths, 496 nm and 565 nm, respectively. This allows for optimal excitation of PE-containing tandem dyes such as PE-Vio615 using blue, green, and yellow green laser lines (488-561 nm). This enables the utilization of the maximum potential of your instrument with flexible choice of laser lines (figure 2). As depicted in figure 1, the extent of absorption at 565 nm is greater than 496 nm and thus, the maximum excitation of PE-Vio615 can be achieved with instruments such the MACSQuant VYB, which uses yellow laser lines for excitation of PE and all PE containing tandem dyes. The emission signal can be detected using typical filters design to detect PE-Texas Red, such as 615/20 nm.

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second dot plot for above text ......

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Enhanced brightness

PE-Vio615 is designed to offer a bright alternative to comparable dyes such as PE-efluor 610, PE-CF594, ECD, and PE/Dazzle 594 (figure 3). PE-Vio615 not only allows for high fluorescent intensities but also excellent separation of positive population for higher stain indices (figure 4). This property of PE-Vio615 makes it an excellent dye for analysis of dim and difficult to characterize markers (figure 5).

ConjugateCloneMFIStain index

MACSQuant VYB

Compensation in

channel Y1 (%)

CD4-PE-Vio615 Vit-4.32363093.0
CD4-ECDSFCl12T4D111331853.3
CD4-PE-594RPA-T42012952.6
CD4-PE-eFluor610RPA-T42022595.5
CD4-PE/Dazzle594RPA-T43162873.9
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second dot plot for above text ......

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third dot plot for above text ......

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Fixation stability

Tandem conjugates are often sensitive to fixatives. Thus, for a successful flow cytomeric analysis, stability of tandem conjugates is critical. PE-Vio615 conjugate shows excellent stability to paraformaldehyde-based fixative without any increase in background signal (table 3).

ConjugateClonewithout fixationwith fixation
MFIStain indexMFIStain index
CD56-PEREA19612.033.211.932.7
CD56-PE-Vio615REA19639.970.940.171.4
CD56-PE/Dazzle594HD5630.542.231.554.0
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Photostability

The stability of PE-Vio615 upon exposure to ambient light (~850 Lux) was analyzed at different time points. Significantly higher mean fluorescence intensities (MFI) and stain indices (SI) for these time points, compared to commercially available alternatives, were also demonstrated (figure 6).

Red Laser (635 nm) -
APC-Vio770™ (Emmax 770 nm)

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

  • 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.

Table 1: Absorption and emission maxima of fluorochromes related to APC-Vio770.
FluorochromeAbsorption max. (nm)Emission max. (nm)
APC-Vio770652775
APC-Cy7650774
APC-Alexa Fluor750 775
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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.

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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.

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

Compensation in

channel R1 (%)

ACD8-APC-Vio77041.452.87.0
ACD8-APC-Cy740.650.611.0
ACD8-APC-H732.245.89.0
BCD8-APC-Vio770
39.457.87.0
BCD8-APC-Cy738.858.511.0
BCD8-APC-H731.251.89.0
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High stability during fixation

APC-Vio770 shows excellent stability after fixation with paraformaldehyde (fig. 4), similar to PE-Vio770.