Fluorescent dyes

MACS Handbook

1 Fluorescent 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 portfolio and allow a greater selection of antibodies for multiparameter cell analysis.
 

1.1 Vio® Dyes

  • Superior mean fluorescence intensity for excellent signal
  • High stain index for clear population discrimination
  • Hassle-free and ideal for multicolor experiments due to low compensation

Fluorochrome specifications

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-Vio 615488 or 561565619B3655–730Y2615/20
PerCP488482675B3655–730N/AN/A
PerCP-Vio 700488482704B3655–730N/AN/A
PE-Vio 770488 or 561565775B4750 LPY4750 LP
APC561 or 635652660R1655-730Y3661/20
APC-Vio 770561 or 635652775R2750 LPY4750 LP
1.1.1 Violet laser (405 nm)

VioBlue® Dye (Emmax 452 nm)

The VioBlue® Dye is a coumarin-based fluorochrome with excitation and emission wavelengths of 400 nm and 455 nm, respectively. It is 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 fluorochromes 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.
 

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 combinations for multiparameter flow cytometry
  • Combined use with the VioGreen Dye possible

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

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

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 (PBMCs) or mouse splenocytes (MS) 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)

The VioGreen™ Dye is a fluorochrome with a large Stokes shift, emitting strong fluorescence at 520 nm upon excitation at 405 nm. It is a non-protein fluorochrome with significantly increased mean fluorescence intensities and higher stain indices compared to related 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 fluorochromes 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.
 

VioGreen Dye at a glance:

  • Large Stokes shift fluorochrome, 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 fluorochrome
  • Combined use with the VioBlue Dye possible
  • Perfectly suited for multiparameter flow cytometry using all current flow cytometers

Absorption and emission maxima of fluorochromes related to VioGreen. 

FluorochromeAbsorption max. (nm)Emission max. (nm)
VioGreen388520
Pacific Orange400551
Krome Orange398528
AmCyan458489
Horizon V500415500

Laser and filter compatibility

With a standard 525/50 nm filter set, VioGreen exhibits a more favorable 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, human and mouse cell populations stained with a VioGreen-conjugated antibody can be easily identified and distinguished from unstained cells.

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Enhanced brightness
Human peripheral blood mononuclear cells (PBMCs) or mouse splenocytes (MS) 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, as 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 (purple), Horizon V500 (black), or Krome Orange (orange).

High stability during paraformaldehyde and ethanol fixation

The VioGreen Dye exhibits strong photostability during paraformaldehyde and ethanol fixation, with only minimal photo-induced degradation.  This highlights the dye’s suitability for use in studies that require 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 extraordinarily bright dye for the FITC channel. The proprietary multimerization technology allows a 4-fold increase in brightness over the traditional FITC dye. The fluorochrome is excited by the blue laser (488 nm) and its emission peak is at 514 nm. Improved brightness in combination with 25% less spillover into the PE channel enables optimal detection of rare surface markers in the FITC channel.
 

VioBright 515 at a glance:
  • Brightest Vio Dye for the blue laser (488 nm)
  • Excellent choice for surface markers, in particular for antigens with low expression levels
  • Spillover into B2/PE-channel reduced by 25%
  • High photostability for immunofluorescence microscopy applications

Absorption and emission maxima of fluorochromes related to VioBright 515. 

FluorochromeAbsorption max (nm)Emission max (nm)
VioBright 515488514
Vio 515488514
VioBright FITC496522
FITC495520
Alexa Fluor 488495519
BD Horizon Brilliant Blue 515490515

Laser and filter compatibility

VioBright 515 can be excited with a blue laser (488 nm) and displays peak excitation and emission at 488 nm and 514 nm, respectively. Its fluorescence signal can be detected with a standard FITC filter, such as the 525/50 nm filter of the MACSQuant Flow Cytometers. Thus, no changes in detection filters or the cytometer itself are required. With VioBright 515 the potential of 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 markers with low expression levels. With stain indices and mean fluorescent intensities (MFI) higher than those of PE, VioBright 515 staining allows for an excellent resolution of positive and negative cell populations.

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Enhanced brightness
Human PBMCs were stained using CD56 antibodies conjugated to FITC, VioBright FITC, VioBright 515, or BB515 and analyzed by flow cytometry using the MACSQuant Analyzer 10. VB: VioBright; BB: Brilliant Blue

MFI and stain indices of CD56 antibodies conjugated to various fluorochromes. 

ConjugateCloneMFIStain indexMACSQuant Analyzer
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 the optimal dye for the analysis of dim markers, such as CD56. In the example shown here, the CD56+CD3+ NKT cell population from whole blood could be analyzed more precisely (right dot plot) than with an alternative bright dye for the same channel (left dot plot).

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Higher specificity
Human whole blood was stained with CD3-VioBlue (clone REA613) and (A) CD56-BB515 or (B) CD56-VioBright 515 (clone REA196).

High stability during fixation

Bright fluorochromes, such as PE and APC, are often sensitive to fixatives. However, for successful flow cytometric analysis of intracellular markers, stability of the fluorochrome conjugates during fixation is essential. VioBright 515 conjugates show excellent stability towards methanol- and paraformaldehyde-based fixatives with only little compromise in brightness.

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Fixation stability
Human PBMCs were stained using a CD56 antibody (clone REA196) conjugated to VioBright 515 or PE. Cells were analyzed by flow cytometry before and after fixation using paraformaldehyde (PFA) and 90% methanol. Analysis was performed using the MACSQuant Analyzer 10.

Stability of CD56-VioBright 515 and CD56-PE towards fixation with PFA or methanol.

  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

Photostability

The stability of VioBright 515 upon exposure to confocal laser light was analyzed at different time points on a Zeiss LSM710 confocal microscope. Compared to a FITC conjugate, significantly higher mean fluorescence intensities (MFI) were demonstrated for the VioBright 515–conjugated antibody at these time points. The VioBright 515 conjugate showed photostability comparable to the Alexa Fluor 488–conjugated antibody, indicating the excellent suitability of VioBright 515 for immunofluorescence microscopy.

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

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Photostability
Human PBMCs were stained with fluorochrome-conjugated CD4 antibodies conjugated to FITC, Alexa Fluor 488, and VioBright 515. Stained cell samples were fixed with paraformaldehyde 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)

Vio® 515 is an organic small-molecule dye, designed for the B1/FITC channel. Compared to FITC, Vio 515 displays an improved brightness and allows the analysis of intracellular markers that were not accessible in this channel before. The dye thus provides more flexibility in multicolor panel design. Vio 515 shows 25% less spillover into the PE channel and facilitates optimal detection of intracellular markers, such as cytokines and transcription factors. The dye is excited by the blue laser (488 nm), and its emission peak is at 514 nm. 
 

Vio 515 at a glance:

  • Brighter alternative to FITC for detection of intracellular markers, such as cytokines and transcription factors
  • Spillover into B2/PE channel reduced by 25%
  • High photostability for immunofluorescence microscopy applications

Absorption and emission maxima of fluorochromes related to Vio 515. 

FluorochromeAbsorption max (nm)Emission max (nm)
VioBright 515488514
Vio 515488514
VioBright FITC496522
FITC495520
Alexa Fluor 488495519
BD Horizon Brilliant Blue 515490515

Laser and filter compatibility

Upon excitation with a blue laser (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 nm filter of the MACSQuant Instruments. Thus, no changes in detection filters or the flow cytometer itself are required. With Vio 515 the potential of 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 and mean fluorescent intensities (MFI) higher than those of 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 Analyzer 10.

MFI and stain indices of CD4 antibodies conjugated to various fluorochromes. 

ConjugateCloneMFIStain indexMACSQuant Analyzer 
compensation in channel B2 (%)
CD4-FITCVIT425.350.06.4
CD4-Alexa Fluor 488VIT435.768.25.6
CD4-Vio 515VIT435.971.24.4

Higher specificity – better resolution

Vio 515 is an optimal reagent for the analysis of intracellular markers such as cytokines. In this example of PFA-fixed human PBMCs, an Anti-IL-5-Vio 515 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 photostability comparable to Alexa Fluor 488, 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.

Vio 515 displayed a significantly higher mean fluorescence intensity (MFI) than FITC 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 paraformaldehyde 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 excited by a blue laser (488 nm). The VioBright Technology allows for an increased number of conjugated FITC molecules per antibody, compared to conventional FITC conjugation. With a brightness similar to PE, VioBright FITC expands the options of multicolor flow cytometry. 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 rare markers and markers with low expression levels
  • Excellent bright alternative to PE for flexible multicolor panel design
  • Signal detectable in the standard FITC channel with peak excitation and emission wavelengths of 496 nm and 522 nm, respectively
  • Works with standard staining protocol

Absorption and emission maxima of fluorochromes related 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 excitation with a blue laser (488 nm), VioBright FITC displays peak absorption and emission at 496 nm and 522 nm, respectively. Its high-intensity fluorescent signal can be detected using a standard FITC filter, such as the 525/50 nm filter of MACSQuant Instruments. Thus, no changes in detection filters or the flow cytometer itself are required when using VioBright FITC instead of FITC or other related fluorochromes. Along with PE, VioBright FITC increases the potential of the blue laser, one of the most common laser lines available for single- or multilaser instruments.

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Laser and filter compatibility
Absorption (top) and emission (bottom) spectra of VioBright FITC compared to FITC, Alexa Fluor 488, and BD Horizon Brilliant Blue 515 (BB515).

Enhanced brightness

VioBright FITC is a superior choice for the detection of markers with low expression levels. With stain indices and mean fluorescence intensities similar to PE, VioBright FITC staining allows for an excellent resolution of positive and negative cell populations.

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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 cell types requires the specific identification of cellular subsets with low frequencies and a clear resolution of the target population. Thus, highly specific antibodies together with bright conjugates are essential for detection of such rare cell populations. VioBright FITC is an exceptionally bright dye for optimal detection of cell subpopulations and their 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), and CD133/1 (clone AC133) antibodies conjugated to either VioBright FITC or PE.

 MFIStain index
ConjugateVioBright FITCPEVioBright FITCPE
CD253.32.05.04.0
CD3354.84.08.511
CD133/13.54.74.74.7

Spillover

Compared to standard FITC, VioBright FITC displays only a minor increase in spillover into the PE channel. Thus, with a miniminal change in compensation settings, VioBright FITC provides the benefit of a brighter dye.

SampleConjugateMFIStain indexMACSQuant Analyzer 10 
compensation in channel B2 (%)
MACSQuant VYB
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 and Vio 615 as the acceptor dye. PE-Vio 615 is optimized for efficient donor-to-acceptor energy transfer, high fluorescence intensity, and low spillover into the detection channel of the donor dye. 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 (561 nm) laser lines for maximum flexibility
  • Excellent brightness for confident detection of dim, rare, and uncharacterized markers
  • Extensive portfolio of PE-Vio 615–conjugated, recombinantly engineered REAfinity™ Antibodies for high reproducibility

Absorption and emission maxima of fluorochromes related to PE-Vio 615.

FluorochromeAborption 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. This allows for optimal excitation of PE-containing tandem dyes, such as PE-Vio 615, by blue, green, and yellow laser lines (488–561 nm). Absorption at 565 nm is greater than at 496 nm. Therefore, maximum excitation of PE-Vio 615 is achieved with instruments such as the MACSQuant VYB, which uses yellow laser lines for excitation of PE and PE-containing tandem dyes. The emission signal can be detected using typical filters designed for PE-Texas Red, such as a 615/20 nm filter.

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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-Vio 615 and analyzed using a MACSQuant VYB (A) and MACSQuant Analyzer (B).

Enhanced brightness

PE-Vio 615 is designed to offer a bright alternative to 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-eFluor 610, PE-CF594, PE-Vio 615, ECD, and PE-Dazzle 594 and analyzed by flow cytometry using the MACSQuant VYB.

MFI and stain indices of CD4 antibodies conjugated to PE-Vio 615, ECD, PE-CF594, PE-eFluor 610, and PE/Dazzle 594.

ConjugateCloneMFIStain indexMACSQuant VYB
compensation in channel Y (%)
     
CD4-PE-Vio 615Vit-4.32363093.0
CD4-ECDSFCI12T4D111331853.3
CD4-PE-CF594RPA-T42012952.6
CD4-PE-eFluor 610RPA-T42022595.5
CD4-PE/Dazzle 594RPA-T43162873.9

PE-Vio 615 allows for high fluorescent intensities and stain indices, enabling excellent distinction between positive and negative cell populations. This makes PE-Vio 615 an optimal dye for the analysis of dim markers and markers that are difficult to characterize.

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Enhanced brightness
Human PBMCs were stained using CD56 antibodies (clone REA196) conjugated to either PE-Vio 615 or PE. Cells were also labeled with  a CD335-VioBright FITC antibody and then analyzed by flow cytometry using the MACSQuant VYB.

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Enhanced brightness
PBMCs were stimulated for 6 hours. After 2 hours, 1 μg/mL BrefeldinA was added for the remaining 4 hours. Cells were stained extracellularly with CD4-FITC, fixed, permeabilized with the Inside Stain Kit (Miltenyi Biotec), and stained intracellularly with CD154-VioBlue and Anti-IL-4-PE or Anti-IL-4-PE-Vio 615 (clone: 7A3-3) for subsequent flow cytometry analysis. Cells were gated on CD4+ lymphocytes. Numbers indicate cell frequencies among CD4+ cells. Data courtesy of Petra Bacher, Clinic for Rheumatology and Clinical Immunology, Charité – University Medicine Berlin, Berlin, Germany.

High stability during fixation

Tandem conjugates are often sensitive to fixatives. However, for many flow cytometric analyses requiring prior fixation, stability of tandem conjugates is critical. PE-Vio 615 shows excellent stability to paraformaldehyde fixation 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-Vio 615, or PE-Dazzle 594. In addition, cells were stained with CD335-VioBright FITC and analyzed before and after fixation using paraformaldehyde. Stained cells were analyzed by flow cytometry.

Photostability

The stability of PE-Vio 615 upon exposure to ambient light was analyzed at different time points. PE-Vio 615 showed significantly higher mean fluorescence intensities (MFI) and stain indices at these time points than commercially available alternatives.

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Photostability
Human PBMCs were stained with CD56 antibodies conjugated to PE-Vio 615 (clone REA196) or PE-Dazzle 594 (clone HCD56). Stained cells were then exposed to ambient light (~850 lux) and analyzed at different time points by flow cytometry using the MACSQuant VYB. Spillover into the Y1 channel, which is optimized for detection of the PE signal, was also analyzed after exposure to light.

PerCP-Vio® 700 (Emmax 704 nm)

PerCP-Vio® 700 is a tandem conjugate that combines the peridinin chlorophyll protein (PerCP) and the Vio 700 dye to emit strong fluorescence at 655–730 nm upon excitation with a blue laser at 488 nm. This dye is suited perfectly for the B3 channel of the MACSQuant Analyzer.

PerCP-Vio 700 at a glance:

  • Optimal excitation with blue laser (488 nm) 
  • Emits strong fluorescence at 655–730 nm
  • Extensive portfolio of PerCP-Vio 700–conjugated, recombinantly engineered REAfinity Antibodies for high reproducibility

Absorption and emission maxima of fluorochromes related to PerCP-Vio 700.

FluorochromeAbsorption max. (nm)Emission max. (nm)
PerCP482675
PerCP-Vio 700482704
PerCP-Cy5.5490695

Laser and filter compatibility

When used in combination with a standard 655–730 nm bandpass filter, PerCP-Vio 700 exhibits a very narrow emission spectrum 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-Vio 700. The blue box represents the 655–730 nm filter.

Enhanced brightness

Human peripheral blood mononuclear cells and mouse splenocytes were stained with various antibodies conjugated to PerCP-Vio 700. Excellent distinction between positive and negative cell populations was observed for many different antibody specificities.

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Enhanced brightness
Human peripheral blood mononuclear cells (PBMCs) or mouse splenocytes (MS) were stained with CD20-PerCP-Vio 700 (A; PBMCs), CD4-PerCP-Vio 700 (B; PBMCs), CD45-PerCP-Vio 700 (C; PBMCs) or CD90.2-PerCP-Vio 700 (D; MS) and analyzed by flow cytometry using the MACSQuant Analyzer.

High stability during fixation

PerCP-Vio 700 shows excellent stability towards fixatives, with only minimal decrease in fluorescence after fixation.

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Fixation stability
CD8-PerCP-Vio 700 before (A) and after (B) incubation with 3.7% paraformaldehyde, indicating only a minimal decrease in fluorescence after fixation.

Photostability

Analysis of the photostability of CD14-PerCP-Vio 700 indicated no discernable changes after up to four hours of continuous exposure to ambient light (~850 lux). PerCP-Vio 700 showed significantly higher mean fluorescence intensities (MFI) and stain indices at these time points than commercially available alternatives.

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Photostability
Antibodies conjugated to PerCP, PerCP‑Vio 700, and PerCP-Cy 5.5 were exposed to ambient light for up to four hours and showed excellent photostability over this time course.

MarkerCloneStain index  
  t0 mint120 mint240 min
CD14-PerCPTÜK4534436
CD14-PerCP-Vio 700TÜK4757575
CD14-PerCP-Cy 5.561D3363128

PE-Vio® 770 (Emmax 770nm)

PE-Vio® 770 is a tandem conjugate, like PE-Cy7, which is based on 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 (Vio 770) dye.
 

PE-Vio 770 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 Vio 770 as the acceptor dye and an optimized chemistry gives rise to a tandem dye that is characterized by a high fluorescence intensity, minimal spillover into adjacent detection channels, and low non-specific binding to non-target cells to meet the complexity of multiparameter flow cytometry
  • Higher mean fluorescence intensity (MFI) and stain index values, in addition to lower compensation requirements compared to PE-Cy7

Absorption and emission maxima of fluorochromes related to PE-Vio 770.

FluorochromeAbsorption max. (nm)Emission max. (nm)
PE-Vio 770565775
PE-Cy7496774
PE-Alexa Fluor 750496775

Laser and filter compatibility

The tandem dyes PE-Vio 770, 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 suited to provide maximum excitation of the PE molecules, which in turn transfer this energy to the respective acceptor molecule. Vio 770 shows emission properties similar to Cy7 and Alexa Fluor 750.

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Laser and filter compatibility
Absorption and emission spectra of PE-Vio 770.

Enhanced brightness

PE-Vio 770 provides the greatest fluorescence intensity of the Vio Dye family, with excellent distinction between positive and negative cells. 

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

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 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-Vio 770 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-Vio 770 and CD8-PE-Cy7.

SampleConjugateMFIStain indexCompensation in channel B2 (%)
ACD8-PE-Vio 770109.297.30.4
ACD8-PE-Cy767.072.72.2
BCD8-PE-Vio 770101.0112.00.4
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-Vio 770 shows excellent stability as shown below.

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Fixation stability
CD45RA-PE-Vio 770 fluorescence before (A) and after (B) 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 and Vio 615 as the acceptor dye. PE-Vio 615 is optimized for efficient donor-to-acceptor energy transfer, high fluorescence intensity, and low spillover into the detection channel of the donor dye. 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 (561 nm) laser lines for maximum flexibility
  • Excellent brightness for confident detection of dim, rare, and uncharacterized markers
  • Extensive portfolio of PE-Vio 615–conjugated, recombinantly engineered REAfinity Antibodies for high reproducibility

Absorption and emission maxima of fluorochromes related to PE-Vio 615.

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. This allows for optimal excitation of PE-containing tandem dyes, such as PE-Vio 615, by blue, green, and yellow laser lines (488–561 nm). Absorption at 565 nm is greater than at 496 nm. Therefore, maximum excitation of PE-Vio 615 is achieved with instruments such as the MACSQuant VYB, which uses yellow laser lines for excitation of PE and PE-containing tandem dyes. The emission signal can be detected using typical filters designed for PE-Texas Red, such as a 615/20 nm filter.

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Laser and filter compatibility
Absorption (A) and emission (B) spectra of PE-Vio 615 and related fluorochromes.

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Laser and filter compatibility
Human PBMCs were stained using CD4 antibodies (clone VIT4) conjugated to PE-Vio 615 and analyzed using a MACSQuant VYB (A) and MACSQuant Analyzer (B).

Enhanced brightness

PE-Vio 615 is designed to offer a bright alternative to 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-eFluor 610, PE-CF594, PE-Vio 615, ECD, and PE-Dazzle 594 and analyzed by flow cytometry using the MACSQuant VYB.

MFI and stain indices of CD4 antibodies conjugated to PE-Vio 615, ECD, PE-CF594, PE-eFluor 610, and PE/Dazzle 594.

ConjugateCloneMFIStain indexMACS Quant VYB 
compensation in channel Y1 (%)
CD4-PE-Vio 615Vit-4.32363093.0
CD4-ECDSFCI12T4D111331853.3
CD4-PE-CF594RPA-T42012962.6
CD4-PE-eFluor 610RPA-T42022595.5
CD4-PE/Dazzle 594RPA-T43162873.9

PE-Vio 615 allows for high fluorescent intensities and stain indices, enabling excellent distinction between positive and negative cell populations. This makes PE-Vio 615 an optimal dye for the analysis of dim markers and markers that are difficult to characterize.

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Enhanced brightness
Human PBMCs were stained using CD56 antibodies (clone REA196) conjugated to either PE-Vio 615 or PE. Cells were also labeled with  a CD335-VioBright FITC antibody and then analyzed by flow cytometry using the MACSQuant VYB.

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Enhanced brightness
PBMCs were stimulated for 6 hours. After 2 hours, 1 μg/mL BrefeldinA was added for the remaining 4 hours. Cells were stained extracellularly with CD4-FITC, fixed, permeabilized with the Inside Stain Kit (Miltenyi Biotec), and stained intracellularly with CD154-VioBlue and Anti-IL-4-PE or Anti-IL-4-PE-Vio 615 (clone: 7A3-3) for subsequent flow cytometry analysis. Cells were gated on CD4+ lymphocytes. Numbers indicate cell frequencies among CD4+ cells. Data courtesy of Petra Bacher, Clinic for Rheumatology and Clinical Immunology, Charité – University Medicine Berlin, Berlin, Germany.

High stability during fixation

Tandem conjugates are often sensitive to fixatives. However, for many flow cytometric analyses requiring prior fixation, stability of tandem conjugates is critical. PE-Vio 615 shows excellent stability to paraformaldehyde fixation 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-Vio 615, or PE-Dazzle 594. In addition, cells were stained with CD335-VioBright FITC and analyzed before and after fixation using paraformaldehyde. Stained cells were analyzed by flow cytometry.
 

Photostability

The stability of PE-Vio 615 upon exposure to ambient light was analyzed at different time points. PE-Vio 615 showed significantly higher mean fluorescence intensities (MFI) and stain indices at these time points than commercially available alternatives.

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Photostability
Human PBMCs were stained with CD56 antibodies conjugated to PE-Vio 615 (clone REA196) or PE-Dazzle 594 (clone HCD56). Stained cells were then exposed to ambient light (~850 lux) and analyzed at different time points by flow cytometry using the MACSQuant VYB. Spillover into the Y1 channel, which is optimized for detection of the PE signal, was also analyzed after exposure to light.

PE-Vio® 770  (Emmax 770 nm)

PE-Vio® 770 is a tandem conjugate, like PE-Cy7, which is based on 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 (Vio 770) dye.
 

PE-Vio 770 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 Vio 770 as the acceptor dye and an optimized chemistry gives rise to a tandem dye that is characterized by a high fluorescence intensity, minimal spillover into adjacent detection channels, and low non-specific binding to non-target cells to meet the complexity of multiparameter flow cytometry
  • Higher mean fluorescence intensity (MFI) and stain index values, in addition to lower compensation requirements compared to PE-Cy7

Absorption and emission maxima of fluorochromes related to PE-Vio 770.

FluorochromeAbsorption max. (nm)Emission max. (nm)
PE-Vio 770565775
PE-Cy7496774
PE-Alexa Fluor 750496775

Laser and filter compatibility

The tandem dyes PE-Vio 770, 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 suited to provide maximum excitation of the PE molecules, which in turn transfer this energy to the respective acceptor molecule. Vio 770 shows emission properties similar to Cy7, and Alexa Fluor 750.

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Laser and filter compatibility
Absorption and emission spectra of PE-Vio 770.

Enhanced brightness

PE-Vio 770 provides the greatest fluorescence intensity of the Vio Dye family, with excellent distinction between positive and negative cells. 
 

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

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-Vio 770 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-Vio 770 and CD8-PE-Cy7.

SampleConjugateMFIStain indexCompensation in channel B2 (%)
ACD8-PE-Vio 770109.297.30.4
ACD8-PE-Cy767.072.22.2
BCD8-PE-Vio 770101.0112.00.4
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-Vio 770 shows excellent stability as shown below.

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Fixation stability
CD45RA-PE-Vio 770 fluorescence before (A) and after (B) 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-Vio® 770 (Emmax 770 nm)

APC-Vio® 770 dye is a tandem conjugate, like APC-Cy7 or APC-H7, which is based on 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 (Vio 770).
 

APC-Vio 770 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 Vio 770 as the acceptor dye and an optimized chemistry gives rise to 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 mean fluorescence intensity (MFI) and stain index values, in addition to lower compensation requirements compared to APC-Cy7

Absorption and emission maxima of fluorochromes related to APC-Vio 770.

FluorochromeAbsorption max. (nm)Emission max. (nm)
APC-Vio 770652775
APC-Cy7650774
APC-Alexa Fluor 750650775

Laser and filter compatibility

The tandem dyes APC-Vio 770, APC-Cy7, and APC-H7 all use APC as the donor fluorochrome, showing maximum absorption around 652 nm. Emission spectra for Vio 770, Cy7, H7, and Alexa Fluor 750 are similar. Therefore, APC-Vio 770 is an ideal tandem conjugate for this channel in all flow cytometers.

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Laser and filter compatibility
Absorption and emission spectra of APC-Vio 770.

Enhanced brightness

APC-Vio 770 provides strong staining, allowing the identification and analysis of specific cell populations .
 

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Enhanced brightness
The strong staining pattern of APC-Vio 770 is illustrated by comparing unstained (left), CD16-APC-stained (middle), and CD16-APC-Vio 770-stained (right) cellular material, after gating on leukocytes and dead cell exclusion.

Compared to other spectrally similar conjugates, such as APC-Cy7 and APC-H7, APC-Vio 770 exhibits equal or stronger staining patterns. In addition, APC-Vio 770 generally shows 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. These properties make APC-Vio 770 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-Vio 770, 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-Vio 770, CD8-APC-Cy7, and CD8-APC-H7.

SampleConjugateMFIStain indexCompensation in channel R1 (%)
ACD8-APC-Vio 77041.452.87.0
ACD8-APC-Cy740.650.611.0
ACD8-APC-H732.245.89.0
BCD8-APC-Vio 77039.457.87.0
BCD8-APC-Cy738.858.511.0
BCD8-APC-H731.251.89.0

High stability during fixation

APC-Vio 770 shows excellent stability after fixation with paraformaldehyde, similar to PE-Vio 770.

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Fixation stability
Cells were stained with CD45-APC-Vio 770 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.