MACS Handbook

Fluorescence dyes

1 Fluorescence dyes

Flow cytometry has an inherent need to continuously increase the number of fluorochromes to meet the
growing demand of sophisticated multicolor analysis 

Vio® Dyes represent a family of fluorochromes for flow cytometry and fluorescence microscopy developed
by Miltenyi Biotec. VioBlue®, VioGreen™, VioBright™ FITC, PE-Vio® 615, PE-Vio® 770, PerCP-Vio® 700, Vio® 515, VioBright™515, APC-Vio® 770 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 Vio Dyes expand Miltenyi Biotec’s antibody offering an allow a greater selection of antibodies for multiparameter cell analysis.

1.1 Vio dyes

  • Bright: superior mean fluorescence intensity for excellent signal
  • Distinct: high stain index for clear population discriminatio
  • Hassle-free: ideal for multicolor experiments due to low compensation
Exitation and emission data of the Vio Dyes
FluorochromeExcitationExmax (nm)Emmax (nm)MACSQuant AnalyzerMACSQuant VYB
laser (nm)ChannelFilter (nm)ChannelFilter (nm)
VioBlue405400452V1450/50V1450/50
VioGreen405388520V2525/50V2525/50
VioBright 515488488514B1525/50B1525/50
Vio 515488488514B1525/50B1525/50
VioBright FITC488496522B1525/50B1525/50
FITC488495520B1525/50B1525/50
PE488 or 561565578B2585/40Y1586/15
PE-Vio615488 or 561565619B3655-730Y2615/20
PerCP488482675B3655-730N/AN/A
PerCP-Vio700488482704B3655-730N/AN/A
PE-Vio770488 or 561565775B4750 LPY4750 LP
APC561 or 635652660R1655-730Y3661/20
APC-Vio770561 or 635652775R2750 LPY4750 LP
1.1.1 Violet laser (405 nm)

VioBlue® Dye (Emmax 452 nm)

VioBlue is Coumarin-based dye with excitation and emission wavelengths of 400 nm and 455 nm, respectively. As a 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. 

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: Absorption and emission maxima of fluorochromes related to VioBlue. 
FluorochromeAbsorption max. (nm)Emission max. (nm)
VioBlue

400 

455
Pacific Blue405455
Cascade Blue(375); 401423
Alexa Fluor 405405421
eFluor 405405450
BD Horizon V450404448
  • 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

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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Laser and filter compatibility
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.

Enhanced brightness

When compared to well-established fluorochromes with high fluorescence intensities, such as PE, VioBlue exhibits a similar degree of fluorescence.

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Enhanced brightness
Human peripheral blood mononuclear cells (PBMC) or mouse splenocyte (MS) cells were stained with CD20-VioBlue (A; PBMCs), the rare cell marker CD123‑VioBlue (B; PBMCs), CD8-VioBlue (C; PBMCs), or CD90.2-VioBlue (D; MS) and analyzed by flow cytometry using the MACSQuant Analyzer.

VioBlue Conjugates provide a superior alternative to many spectrally similar conjugates for the V1 channel, further increasing the options of multicolor analysis.

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Enhanced brightness
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

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, equal to or exceeding many other spectrally similar fluorochromes.

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High stability during fixation
CD123-VioBlue staining before (left) and after (right) fixation with 3.7% paraformaldehyde indicating only a very small decrease in fluorescence after fixation.


VioGreen™ Dye (Emmax 520 nm)


VioGreen is a large stokes shift fluorophore, emitting strong fluorescence at 520 nm upon excitation at 405 nm. It is a non-protein fluorophore with significantly increased mean fluorescence intensities and higher stain indices than comparable fluorochromes such as Pacific Orange™, Krome Orange™, and BD Horizon™ V500. 

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

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

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Laser and filter compatibility
Absorption (top) and emission (bottom) spectra of VioGreen, Pacific Orange, and AmCyan. The blue box represents the 525/50 nm filter.

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 (left), PE- (middle), or VioGreen-labeled (right) cells:

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Enhanced brightness
Human peripheral blood mononuclear cells (PBMC) or mouse splenocyte (MS) cells were stained with CD14-VioGreen (A; PBMCs), CD20-VioGreen (B; PBMCs), CD15-VioGreen (C; PBMCs), or CD8a-VioGreen (D; MS) and analyzed by flow cytometry using the MACSQuant® Analyzer.

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

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-Pacific Orange6.17.0
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Enhanced brightness
Analysis of human PBMCs using CD3 antibodies conjugated to either VioGreen, Horizon V500, or Krome Orange.

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. It is also suitable for ethanol fixation.

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High stability
CD14-VioGreen staining before (left) and after (right) paraformaldehyde fixation, indicating only a very small decrease in fluorescence after fixation.

1.1.2 Blue Laser (488 nm)

VioBright™ 515 (Emmax 514 nm)

VioBright 515 is an extraordinary bright dye for the FITC-channel. The proprietary multimerization technology allows a 4 fold increase in brightness over the traditional FITC dye. The dye is excited by the blue laser (488 nm) and it´s emission peak is at 514 nm. Improved brightness in combination with 25% less spill-over into the PE-channel allows an optimal detection of rare surface markers in the FITC channel.

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
Absorption and emission peaks of fluorochromes compared to VioBright™ 515.
FluorochromeAbsorption max (nm)Emission max (nm)
VioBright 515488514
Vio515488514
VioBright FITC496522
FITC495520
Alexa Fluor 488495519
BD Horizon Brilliant Blue 515490515

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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Laser and filter compatibility
Absorption (top) and emission (bottom) spectra of VioBright™ 515 compared to FITC, VioBright FITC and Vio® 515.

Enhanced brightness

VioBright™ 515 is the best choice for the detection of lowly expressed markers. 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
Human PBMCs were stained using anti-CD56 antibodies conjugated to FITC, VioBright™ FITC, VioBright 515, BB515 and analyzed by flow cytometry using the MACSQuant 10.

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

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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Higher specificity
Human whole blood was stained with CD3(REA613)-VioBlue® and CD56(REA196)-VioBright™ 515 or CD56-BB515, respectively.

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.

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Fixation stability
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.

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

Photo stability

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. VioBright 515 shows comparable stability to Alexa Fluor 488 conjugated antibodies indicating the excellent suitability for immunofluorescence microscopy.

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Photo stability
Human PBMCs were stained with CD4 antibodies conjugated to Alexa Fluor 488 (A) and VioBright 515 (B). Stained cell samples were fixed with p-formaldehyde for 20 min and transferred to a 96-well plate for analysis. Comparable photostability could be observed upon exposure to confocal laser light on a Zeiss LSM710 microscope.

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Photo stability
Human PBMCs were stained with fluorochrome-conjugated CD4 antibodies conjugated to FITC, Alexa Fluor 488 and VioBright 515. Stained cell samples were fixed with p-formaldehyde for 20 min and transferred to a 96-well plate for analysis. The plotted curve is normalized over multiple time points.

Vio® 515 (Emmax 514 nm)

Vio515 is a dye for the FITC-channel. Improved brightness over FITC allows the analysis of intracellular markers that were not available in this channel before, thus giving more flexibility in multicolor panel design. In combination with 25% less spill-over into the PE-channel Vio515 allows an optimal detection of intracellular markers, such as cytokines and transcription factors. The dye is excited by the blue laser (488 nm) and it´s emission peak is at 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
Absorption and emission peaks of fluorochromes compared to Vio® 515.
FluorochromeAbsorption max (nm)Emission max (nm)
VioBright 515488514
Vio515488514
VioBright FITC496522
FITC495520
Alexa Fluor 488495519
BD Horizon Brilliant Blue 515490515

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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Laser and filter compatibility
Absorption (top) and emission (bottom) spectra of Vio®515 compared to FITC, VioBright™ FITC and VioBright 515.

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 the figure below.

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Enhanced brightness
Human PBMCs were stained using CD4 (clone VIT4) antibodies conjugated to FITC, Vio® 515 and Alexa Fluor 488. Samples were analyzed by flow cytometry using the MACSQuant®10.

ConjugateCloneMFIStain indexMACS Quant 
Compensation in Channel B2 [%]
CD4-FITCVIT425.350.06.4
CD4-Alexa488VIT435.768.25.6
CD4-Vio515VIT435.971.24.4

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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Higher specificity
Human peripheral blood mononuclear cells (PBMCs) were either left unstimulated (A) or stimulated (B) with PMA/ionomycin for six hours. After two hours, brefeldin A was added. The cells were fixed, permeabilized, and intracellularly stained with Anti-IL-5 antibodies. Cells were then analyzed by flow cytometry. Plots show CD4+ lymphocytes gated according to CD4-APC staining.

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.

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Photostability
Human PBMCs were stained with CD4 antibodies conjugated to Alexa Fluor 488 (A) and Vio 515 (B). Stained cell samples were fixed with p-formaldehyde for 20 min and transferred to a 96-well plate for analysis. Comparable photostability could be observed upon exposure to confocal laser light on a Zeiss LSM710 microscope.

A significantly higher mean fluorescence intensity (MFI) compared to FITC conjugate antibodies, was demonstrated at different time points.

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Photostability
Human PBMCs were stained with different fluorochrome-conjugated CD4 antibodies. Stained cell samples were fixed with p-formaldehyde for 20 min and transferred to a 96-well plate for analysis. The plotted curve is normalized over multiple time points.

VioBright™ FITC (Emmax 522 nm)

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
Absorption and emission maximums of fluorochromes comparable to VioBright FITC
FluorochromeAbsorption max. (nm)Emission max. (nm)
VioBright FITC496522
FITC495520
Alexa Fluor 488495519
BD Horizon Brilliant Blue 515490515

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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Laser and filter compatibility
Absorption (top) and emission (bottom) spectra of VioBright™ FITC compared to FITC, Alexa® 488 and BD Horizon Brilliant™ Blue 515

Enhanced brightness

VioBright FITC is a superior choice of conjugate for detection of low-expressed markers, as shown  below. 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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Enhanced brightness
Detection of CD25+ cells using VioBright FITC conjugated antibodies.

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Enhanced brightness
Detection of CD335+ cells using VioBright FITC conjugated antibodies.

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, VioBright FITC offers you the bright dye alternative for optimal detection of your subpopulation of interest and detailed phenotypic analysis.

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Enhanced brightness
Detection of CD133/1+ cells using VioBright FITC conjugated antibodies

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

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

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-Vio 615 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
FluorochromeAborption max. (nm)Emission max. (nm)
PE-Vio615565619
ECD, PE-Texas Red565613
PE-efluor 610565606
PE-CF594565614
PE/Dazzle 594566612

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. As depicted , 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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Laser and filter compatibility
Absorption (A) and emission (B) spectra of PE-Vio615

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Laser and filter compatibility
Human PBMCs were stained using CD4 antibodies (clone VIT4) conjugated to PE-Vio615 followed by analysis using MACSQuant VYB (A) and MACSQuant Analyzer (B).

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. 

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Enhanced brightness
Human PBMCs were stained using CD4 antibodies conjugated to PE-eFluor610, PE-CF594, PE-Vio615, ECD, PE-Dazzle594 and analyzed by flow cytometry using the MACSQuant VYB.

MFI and stain indices of CD4 antibodies conjugated to PE-Vio615, ECD, PE-CF594, PE-eFluor610, PE/Dazzle594.
ConjugateCloneMFIStain indexMACS Quant VYB
Compensation in channel Y (%)
CD4-PE-Vio615Vit-4.32363093.0
CD4-ECDSFCI12T4D111331853.3
CD4-PE-CF594RPA-T42012952.6
CD4-PE-efluor610RPA-T42022595.5
CD4-PE/Dazzle594RPA-T43162873.9
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).
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Enhanced brightness
Human PBMCs were stained using CD56 antibodies (clone REA196) conjugated to either PE-Vio615 or PE and CD335-VioBright FITC. Stained cells were then analyzed by flow cytometry using the MACSQuant VYB.

This property of PE-Vio615 makes it an excellent dye for analysis of dim and difficult to characterize markers.
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Enhanced brightness
PBMCs were stimulated for 6 hours. After 2 hours, 1 μg/mL BrefeldinA was added for the last 4 hours. Cells were surface stained with CD4-FITC, fixed and permeabilized with the Inside Stain Kit (Miltenyi Biotec) and stained intracellularly with CD154-VioBlue and Anti-IL-4-PE or Anti-IL-4-PE-Vio615 (clone: 7A3-3). Cells were acquired on a BD Fortessa. Cells are gated on CD4+ lymphocytes, numbers indicate frequency among CD4+ cells. Data courtesy: Petra Bacher, Clinic for Rheumatology and Clinical Immunology, Charité – University Medicine Berlin, Berlin, Germany

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.

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Fixation stability
Human PBMCs were stained using CD56 antibodies (clone REA196) conjugated to PE, PE-Vio615 or PE-Dazzle594. In addition, cells were stained with CD335-VioBright FITC and analyzed before and after fixation using paraformaldehyde. Stained cells were analyzed by flow.

Photo stability

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.

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Photo stability
Human PBMCs were stained, using CD56 antibodies conjugated to PE-Vio615 (clone REA196) or PE-Dazzle594 (clone HCD56). Stained cells were then exposed to ambient light (~850 Lux) followed by analysis at different time points by flow cytometry using the MACSQuant VYB. Spill over in the Y1 channel, which is optimized for detection of PE signal was also analyzed after exposure to light.

PerCP-Vio700 Dye (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:

Table 1: Absorption and emission maxima of fluorochromes related to PerCP-Vio700.
FluorochromeAbsorption max. (nm)Emission max. (nm)
PerCP482675
PerCP-Vio700482704
PerCp-Cy5.5490695

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.

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Laser and filter compatibility
Absorption and emission spectra of PerCP-Vio700. The blue box represents the 655–730 filter.

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.

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Enhanced brightness
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.

High stability during fixation

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

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Fixation stability
CD8-PerCP-Vio700 before (A) and after (B) fixation with 3.7% paraformaldehyde, indicating a minimal decrease in fluorescence after fixation.

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.

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Photo stability
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.

MarkerCloneStain index
t0 mint120 mint240 min
CD14-PerCPTÜK4534436
CD14-PerCP-Vio700TÜK4757575
CD14-PerCP-Cy5.561D3363128

PE-Vio™ 770 Dye (Emmax 770nm)

The PE-Vio®770 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.
Absorption and emission maxima of fluorochromes related to PE-Vio770.
FluorochromeAbsorption max. (nm)Emission max. (nm)
PE-Vio770565775
PE-Cy7496774
PE-Alexa Fluor 750496775

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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Laser and filter compatibility
Absorption and emission spectra of PE-Vio770.

Enhanced brightness

PE-Vio 770 provides the greatest fluorescence intensity of the Vio Dye family, with excellent separation of positively and negatively stained cells. When compared to other spectrally similar tandem conjugates, such as PE-Cy7 (figure 31) or PE-Alexa Fluor 750, PE-Vio 770 exhibits significantly higher mean fluorescence intensities (MFI) and stain indices, and requires less compensation. These properties make PE-Vio 770 a far superior tandem conjugate compared to PE-Cy7 or PE-Alexa Fluor 750.

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Enhanced brightness
Human peripheral blood mononuclear cells (PBMC) or mouse splenocyte (MS) cells were stained with CD3-PE-Vio 770 (A; PBMCs), CD14-PE-Vio 770 (B; PBMCs), CD19-PE-Vio770 (C; PBMCs), or CD4-Biotin/Anti-Biotin-PE-Vio 770 (D; MS) and analyzed by flow cytometry using the MACSQuant Analyzer. 

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Enhanced brightness
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.

MFI, stain indices, and compensation requirements of CD8-PE-Vio770 and CD8-PE-Cy7.
SampleConjugateMFIStain indexCompensation 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

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 below.

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Fixation stability
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.

1.1.3 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-Vio 615 dye conjugated to recombinantly engineered REAfinity Antibodies for higher reproducibility.
Absorption and emission maximums of fluorochromes comparable to PE-Vio615
FluorochromeAbsorption max. (nm)Emission max. (nm)
PE-Vio 615565619
ECD, PE-Texas Red565613
PE-efluor 610565606
PE-CF594565614
PE/Dazzle 594566612
 

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. As depicted, 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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Laser and filter compatibility
Absorption (A) and emission (B) spectra of PE-Vio 615

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Laser and filter compatibility
Human PBMCs were stained using CD4 antibodies (clone VIT4) conjugated to PE-Vio615 followed by analysis using MACSQuant VYB (A) and MACSQuant Analyzer (B).

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. 

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Enhanced brightness
Human PBMCs were stained using CD4 antibodies conjugated to PE-eFluor610, PE-CF594, PE-Vio615, ECD, PE-Dazzle594 and analyzed by flow cytometry using the MACSQuant VYB.

MFI and stain indices of CD4 antibodies conjugated to PE-Vio615, ECD, PE-CF594, PE-eFluor610, PE/Dazzle594.
ConjugateCloneMFIStain indexMACS Quant VYB Compensation in channel Y1 (%)
CD4-PE-Vio615Vit-4.32363093.0
CD4-ECDSFCI12T4D111331853.3
CD4-PE-CF594RPA-T42012962.6
CD4-PE-eFluor610RPA-T42022595.5
CD4-PE/Dazzle594RPA-T43162873.9
PE-Vio615 not only allows for high fluorescent intensities but also excellent separation of positive population for higher stain indices.
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Enhanced brightness
Human PBMCs were stained using CD56 antibodies (clone REA196) conjugated to either PE-Vio615 or PE and CD335-VioBright FITC. Stained cells were then analyzed by flow cytometry using the MACSQuant VYB.

This property of PE-Vio615 makes it an excellent dye for analysis of dim and difficult to characterize markers.
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Enhanced brightness
PBMCs were stimulated for 6 hours. After 2 hours, 1 μg/mL BrefeldinA was added for the last 4 hours. Cells were surface stained with CD4-FITC, fixed and permeabilized with the Inside Stain Kit (Miltenyi Biotec) and stained intracellularly with CD154-VioBlue and Anti-IL-4-PE or Anti-IL-4-PE-Vio615 (clone: 7A3-3). Cells were acquired on a BD Fortessa. Cells are gated on CD4+ lymphocytes, numbers indicate frequency among CD4+ cells. Data courtesy: Petra Bacher, Clinic for Rheumatology and Clinical Immunology, Charité – University Medicine Berlin, Berlin, Germany

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).

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Fixation stability
Human PBMCs were stained using CD56 antibodies (clone REA196) conjugated to PE, PE-Vio615 or PE-Dazzle594. In addition, cells were stained with CD335-VioBright FITC and analyzed before and after fixation using paraformaldehyde. Stained cells were analyzed by flow cytometry.

Photo stability

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:

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Photo stability
Human PBMCs were stained, using CD56 antibodies conjugated to PE-Vio615 (clone REA196) or PE-Dazzle594 (clone HCD56). Stained cells were then exposed to ambient light (~850 Lux) followed by analysis at different time points by flow cytometry using the MACSQuant VYB. Spill over in the Y1 channel, which is optimized for detection of PE signal was also analyzed after exposure to light.

PE-Vio770™ Dye (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.
Table 1: Absorption and emission maxima of fluorochromes related to PE-Vio770.
FluorochromeAbsorption max. (nm)Emission max. (nm)
PE-Vio770565775
PE-Cy7496774
PE-Alexa Fluor 750496775

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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Laser and filter compatibility
Absorption and emission spectra of PE-Vio770.

Enhanced brightness

PE-Vio 770 provides the greatest fluorescence intensity of the Vio Dye family, with excellent separation of positively and negatively stained cells.

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Enhanced brightness
Human peripheral blood mononuclear cells (PBMC) or mouse splenocyte (MS) cells were stained with CD3-PE-Vio 770 (A; PBMCs), CD14-PE-Vio 770 (B; PBMCs), CD19-PE-Vio770 (C; PBMCs), or CD4-Biotin/Anti-Biotin-PE-Vio 770 (D; MS) and analyzed by flow cytometry using the MACSQuant Analyzer. 

 When compared to other spectrally similar tandem conjugates, such as PE-Cy7  or PE-Alexa Fluor 750, PE-Vio 770 exhibits significantly higher mean fluorescence intensities (MFI) and stain indices, and requires less compensation. These properties make PE-Vio 770 a far superior tandem conjugate compared to PE-Cy7 or PE-Alexa Fluor 750.
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Enhanced brightness
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.

MFI, stain indices, and compensation requirements of CD8-PE-Vio770 and CD8-PE-Cy7.
SampleConjugateMFIStain indexCompensation in channel BS (%)
ACD8-PE-Vio770109.297.30.4
ACD8-PE-Cy767.072.22.2
BCD8-PE-Vio770101.0112.00.4
BCD8-PE-Cy765.681.72.2

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 below.

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Fixation stability
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.

1.1.4 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.
Absorption and emission maxima of fluorochromes related to APC-Vio770.
FluorochromeAbsorption max. (nm)Emission max. (nm)
APC-Vio770652775
APC-Cy7650774
APC-Alexa Fluor 750650775

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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Laser and filter compatibility
Absorption and emission spectra of APC-Vio770.

Enhanced brightness

APC-Vio770 provides strong staining, allowing the identification and analysis of specific cell populations .

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Enhanced brightness
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.

When compared to other spectrally similar conjugates, such as APC-Cy7 and APC-H7, APC-Vio770 exhibits equal or stronger staining patterns. 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.

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Enhanced brightness
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.

MFI, stain indices, and compensation requirements of CD8-APC-Vio770, CD8-APC-Cy7, and CD8-APC-H7.
SampleConjugateMFIStain indexCompensation in channel R1 (%)
ACD8-APC-Vio77041.452.87.0
ACD8-APC-Cy740.650.611.0
ACD8-APC-H732.245.89.0
BCD8-APC-Vio77039.457.87.0
BCD8-APC-Cy738.858.511.0
BCD8-APC-H731.251.89.0

High stability during fixation

APC-Vio770 shows excellent stability after fixation with paraformaldehyde, similar to PE-Vio770.

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Fixation stability
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.