Watch your multicolor panels grow with Spark Dyes, a family of small, synthetic fluorophores. The addition of these expertly-crafted dyes to BioLegend’s portfolio provides researchers with the ability to fill spectral spaces between existing fluorophores, maximizing panel flexibility. Spark Dyes are advantageous due to their relatively narrow emission profile, stability, and solubility. In addition, their synthetic nature means they are not typically sensitive to standard fixatives, including organic solvents used for phospho-flow. Learn more about each of our Spark Dyes and how you can best incorporate them in multicolor applications.

Spark Violet™ 538

 

Spark Violet™ 538 slots in as an additional violet laser fluor option for multicolor flow cytometry users. This dye is optimally excited by the 405 nm violet laser and emits maximally at 538 nm, placing its peak emission between Brilliant Violet 510™ and Brilliant Violet 570™. When used on a spectral cytometer, Spark Violet™ 538 can be successfully unmixed from neighboring dyes, such as BV510™, BV570™, and BV605™. While it is brighter than Pacific Orange™, Spark Violet™ 538 is relatively dim and should be paired with antigens expressed at high levels.

 

 

Excitation and Emission Spectra of Spark Violet™ 538

 

Spark Violet 538 Spectra Signature

 

Emission spectra (top) and normalized emission spectra (middle) of Spark Violet™ 538 run on a 5-laser Cytek™ Aurora Spectral Cytometer. To compare Spark Violet™ 538 with other fluorophores on a spectral cytometer, use our Aurora Spectral Analyzer tool.

 

Normalized excitation and emission spectra of Spark NIR™ 685 obtained from a spectrophotometer (bottom). To compare Spark Violet™ 538 with other fluorophores, use our Fluorescence Spectra Analyzer tool.

A Perfect Complement to Brilliant Violet™ Dyes

In the data below, Spark Violet™ 538 is utilized with several neighboring Brilliant Violet™ dyes, showcasing its ability to be spectrally unmixed from these dyes in a large, multicolor panel. 

 

Spark Violet 538 panel

 

Lysed human whole blood was stained with the indicated antibodies in the presence of Brilliant Stain Buffer. Samples were unmixed on a Cytek™ Aurora Cytometer using compensation beads and cells. All plots are gated on lymphocytes.

 

A New Alternative Pacific Orange™

Spark Violet™ 538 is spectrally similar to Pacific Orange™ with a peak emission in the V8 channel on the Cytek™ Aurora. Spark Violet™ 538 offers a brighter signal when compared to Pacific Orange™ in a side-by-side experiment.

 

Spark Violet 538 Titration

 

(Left) Human lysed whole blood was stained with anti-human CD4, clone SK3, conjugated to either Spark Violet™ 538 (solid purple) or Pacific Orange™ (clone S3.5, clear dashed) and then analyzed on a Cytek™ Aurora cytometer. Plots are gated on lymphocytes. (Right) Titration curve generated by staining human lysed whole blood with Spark Violet™ 538 conjugated to anti-CD4, clone SK3, antibody.  

 

Stability and Validation Testing

 

All BioLegend fluorophores undergo rigorous testing procedures to determine how light, heat, and fixation may affect the performance and ensure they will perform reliably. To compare the signal across different conditions and timepoints, we used the Stain Index (formula below) to measure the relative brightness of the antibody

Spark Violet 538 equation

 

Photostability Testing

 

The photostability of Spark Violet™ 538 was tested in two ways that mimic how an antibody may be exposed to light over the course of an experiment.

 

  1. Antibodies were stored in the dark or exposed to fluorescent lighting. Then, the antibodies were used to stain freshly harvested cell samples and analyzed immediately.
  2. Cells were stained with antibody that had been kept under recommended storage conditions. Prior to analysis, the stained cells were stored in the dark or exposed to fluorescent lighting.

Spark Violet 538

Anti-human CD4 Spark Violet™ 538 (clone SK3) or Pacific Orange™ (clone S3.5) antibodies were stored in a clear vial (dye only) and left exposed to light or protected in the dark, as indicated. Antibodies were stored for the indicated timepoints and then used to stain human lysed whole blood. “Dark” samples were only tested at the 24 hour time point as a control. Samples labeled "dye + cells" contain human lysed whole blood that was stained with the indicated antibody. Stained cells were then left in the light or protected, as indicated. 

 

Heat Stability

 

Spark Violet 538 Heat Stability

Anti-human CD4 antibodies, clone SK3 Spark Violet™ 538 or clone S3.5 Pacific Orange™, were kept at either 4°C or 37°C for seven weeks. The antibodies were then used to stain human lysed whole blood.

 

 

Fixative Stability

 

Spark Violet™ 538 is compatible with BioLegend buffers, but may be sensitive to alcohol-based buffers. The end user will need to test to ensure their antigen's signal is maintained post-fixation.

 

A guide to the fixatives used in this experiment:

 

 

Human PBMCs were stained with anti-human CD4 (clone SK3) conjugated to Spark Violet™ 538 and fixed using the respective protocols for each buffer set. For intracellular staining buffers, additional antibody stains for Granzyme A or FOXP3 were performed. For the alcohols, 90% methanol and 70% ethanol were used. Samples were then transferred into FluoroFix™ Buffer and read on a 5-laser Cytek™ Aurora Cytometer immediately.

Spark Blue™ 550

 

Spark Blue™ 550 provides a unique option for adding an additional fluorophore to a high-parameter (>22 color) multicolor panel on spectral unmixing cytometers like the Cytek™ Aurora or SONY ID7000™ Spectral Cell Analyzer. It is excited off of the 488 nm laser and emits at 540 nm, filling the spectral gap between commonly used fluors like FITC and PE (R-Phycoerythrin). Emission off of the 405 nm violet laser further adds to the accuracy with which it can be unmixed from neighboring fluorophores and does not significantly impact the detection of any violet laser fluorophores. Because Spark Blue™ 550 is a relatively dim fluorophore, it is best used to detect a high abundance antigen.

 

 

Excitation and Emission Spectra of Spark Blue™ 550

 

Spark Blue 550 spectra signature

Spark Blue 550 aurora spectra

Spark Blue 550 spectra

 

Emission spectra (top) and normalized emission spectra (middle) of Spark Blue™ 550 run on a 5-laser Cytek™ Aurora Spectral Cytometer. To compare Spark Blue™ 550 with other fluorophores on a spectral cytometer, use our Aurora Spectral Analyzer tool.

 

Normalized excitation and emission spectra (bottom) of Spark Blue™ 550 obtained from a spectrophotometer. To compare Spark Blue™ 550 with other fluorophores, use our Fluorescence Spectra Analyzer tool.

 

Minimal Spillover Considerations

 

Spark Blue™ 550 does not exhibit high spillover with other fluorophores, making it easy to place into a large panel. It is maximally excited by the 488 nm laser with minimal emission off of the 561 nm laser. While emission off of the 405 nm violet laser allows it to be easily unmixed from neighboring fluors, it does not significantly impact spreading into the violet laser fluors. Spectrally, it falls between the emissions of FITC and PE, but can easily be used in the same panel as these fluors with minimal spillover considerations.

 

Spark Blue 550

Spreading impact of Spark Blue™ 550 into detection channels of a 5-laser Cytek™ Aurora Spectral Cytometer.

 

 

Human whole blood was stained with anti-CD19 Spark Blue™ 550, anti-CD4 BV570™, anti-CD38 PE, and anti-CD40 FITC antibodies. Samples were unmixed on a Cytek™ Aurora Cytometer using compensation beads and cells. All plots are gated on lymphocytes.

 

A Brighter Alternative to Alexa Fluor® 532

 

Spark Blue™ 550 is spectrally similar to Alexa Fluor® 532 with a peak emission in the B3 channel on the Cytek™ Aurora and offers a brighter signal when compared to Alexa Fluor® 532 in a side-by-side experiment. Due to its unique emission peak off of the violet laser, Spark Blue™ 550 is typically easier to unmix from neighboring fluors when compared to Alexa Fluor® 532.

 

Spark Blue 550 compared to Alexa Fluor 532

Human peripheral blood lymphocytes were stained with anti-CD8 Pacific Blue™, and either anti-CD4 (clone SK3) Spark Blue™ 550 (left) or anti-CD4 (clone SK3) Alexa Fluor® 532 (right).

 

Stability and Validation Testing

 

All BioLegend fluorophores undergo rigorous testing procedures to determine how light, heat, and fixation may affect the performance and ensure they will perform reliably. To compare the signal across different conditions and timepoints, we used the Stain Index (formula below) to measure the relative brightness of the antibody. 

Spark 550

Titration of Spark Blue™ 550

Titration curves generated by staining human lysed whole blood with Spark Blue™ 550 conjugated anti-CD4 (SK3), anti-CD3 (UCHT1), and anti-CD19 (HIB19) antibodies.

 

Photostability Testing

 

The photostability of Spark Blue™ 550 was tested in two ways that mimic how an antibody may be exposed to light over the course of an experiment.

 

  1. Antibodies were stored in the dark or exposed to fluorescent lighting. Then, the antibodies were used to stain freshly harvested cell samples and analyzed immediately.
  2. Cells were stained with antibody that had been kept under recommended storage conditions. Prior to analysis, the stained cells were stored in the dark or exposed to fluorescent lighting.

 

Spark Blue™ 550 antibodies are stable when left under fluorescent lighting overnight. When stained cells are stored overnight in either condition, there is an accelerated loss of signal due to the additive oxidative stress from the cells on the reagent.

 

Anti-human CD4 (clone SK3) Spark Blue™ 550 was stored in a clear vial (Ab only) and left exposed to light or protected in the dark, as indicated. Antibodies were stored for the indicated timepoints and then used to stain human lysed whole blood. Samples labeled Ab+ cells contain human lysed whole blood that was stained with anti-human CD4 Spark Blue™ 550. Stained cells were then left in the light or protected, as indicated.

 

Heat Stability

 

Anti-human CD4 (clone SK3) Spark Blue™ 550 was aliquoted and incubated at the indicated temperatures over the course of 17 days. The antibodies were then used to stain human lysed whole blood from a single donor.

 

Fixative Stability

 

Spark Blue™ 550 is compatible with all BioLegend fixation buffers. For each buffer set, fresh fixed samples were tested immediately following staining or stored overnight in Cyto-Last™ Buffer before being read on a cytometer.

 

A guide to the fixatives used in this experiment:

 

 

Spark Blue 550

Human PBMCs were stained with anti-human CD4 (clone SK3) conjugated to Spark Blue™ 550 and fixed using the respective protocols for each buffer set. Fresh samples were fixed and read on a Cytek® Aurora immediately. Overnight samples were fixed and stored overnight in Cyto-Last™ Buffer before reading.

Spark NIR™ 685

 

Spark NIR™ 685 expands your options off of the red laser to build larger multicolor panels. Traditionally, the 633 nm red laser has been limited to only 2-3 fluorophores due to the fluors’ wide emission spectra in the near infrared (NIR) range. With the help of spectral unmixing cytometers, the spectral gap between the emission peaks of APC and Alexa Fluor® 700 can now be used to detect an additional fluorophore. Spark NIR™ 685 fills that spectral space with a maximum emission at 685 nm.

 

 

Excitation and Emission Spectra of Spark NIR™ 685

 

Spark NIR685 Spectra Signature

Spark NIR685 Aurora Spectra

Spark NIR685 Spectra

 

Emission spectra (top) and normalized emission spectra (middle) of Spark NIR™ 685 run on a 5-laser Cytek™ Aurora Spectral Cytometer. To compare Spark NIR™ 685 with other fluorophores on a spectral cytometer, use our Aurora Spectral Analyzer tool.

 

Normalized excitation and emission spectra of Spark NIR™ 685 obtained from a spectrophotometer (bottom). To compare Spark NIR™ 685 with other fluorophores, use our Fluorescence Spectra Analyzer tool.

 

Utilize Spark NIR™ 685 With Other Red Laser Fluorophores

 

Spark NIR™ 685 can be used in the same panel as APC or Alexa Fluor® 647; however, spreading error with these fluors must be considered during panel design and panels should be built to match proper fluor brightness with antigen expression. For example, in an ideal panel, Spark NIR™ 685 and Alexa Fluor® 647 would not be used to detect two co-expressed markers.

 

Spark NIR 685

Spreading impact of Spark NIR™ 685 into detection channels of a 5-laser Cytek™ Aurora Spectral Cytometer.

 

Below, Panel A demonstrates how Spark NIR™ 685 can be used in combination with other red laser fluorophores. Panel B is shown as a reference panel using pre-optimized fluorophores to ensure that staining patterns are similar.

 

Spark NIR 685 panel

 

 

Marker

Panel A Fluorophore

Panel B Fluorophore

CD4

Spark NIR™ 685

FITC

CD8

APC/Fire™ 750

Pacific Blue™

CD19

Alexa Fluor® 700

Alexa Fluor® 700

CD56

APC

PE/Cy7

 

Human whole blood from the same donor was stained with the indicated antibodies in either Panel A or Panel B. Cells were washed and fixed with FluoroFix™ prior to analysis.

 

A Brighter Alternative to Alexa Fluor® 660

 

Spark NIR™ 685 is spectrally similar to Alexa Fluor® 660 with a peak emission in the R3 channel on the Cytek™ Aurora. Spark NIR™ 685 offers a brighter signal when compared to Alexa Fluor® 660 in a side-by-side experiment.

 

Spark NIR 685

 

Human lysed whole blood was stained with either anti-CD4 (clone SK3) or anti-CD27 (O323). Antibodies were conjugated to either Spark NIR™ 685 (red) or Alexa Fluor® 660 (blue).

 

Titration curves generated by staining human lysed whole blood with anti-CD4 (clone SK3) antibodies conjugated either to Spark NIR™ 685 or Alexa Fluor® 660 as indicated.

 

Stability and Validation Testing

 

All BioLegend fluorophores undergo rigorous testing procedures to determine how light, heat, and fixation may affect the performance and ensure they will perform reliably. To compare the signal across different conditions and timepoints, we used the Stain Index (formula below) to measure the relative brightness of the antibody

Spark NIR equation

 

Photostability Testing

 

The photostability of Spark NIR™ 685 was tested in two ways to mimic how an antibody may be exposed to light over the course of an experiment.

 

  1. Antibodies were stored in the dark or exposed to fluorescent lighting. Then, the antibodies were used to stain freshly harvested cell samples and analyzed immediately.
  2. Cells were stained with antibody that had been kept under recommended storage conditions. Prior to analysis, the stained cells were stored in the dark or exposed to fluorescent lighting.

Spark NIR

Anti-human CD4 (clone SK3) Spark NIR™ 685 was stored in a clear vial (Ab only) and left exposed to light or protected in the dark, as indicated. Antibodies were stored for the indicated timepoints and then used to stain human lysed whole blood. Samples labeled Ab+ cells contain human lysed whole blood that was stained with anti-human CD4 Spark NIR™ 550. Stained cells were then left in the light or protected, as indicated.

 

Heat Stability

 

Anti-human CD4 (clone SK3) antibody conjugated to Spark NIR™ 685 was aliquoted and incubated at the indicated temperatures over the course of 17 days. The antibodies were then used to stain human lysed whole blood from a single donor.

 

 

Fixative Stability

 

Spark NIR™ 685 is compatible with all BioLegend fixation buffers. For each buffer set, fresh fixed samples were tested immediately following staining or stored overnight in Cyto-Last™ Buffer before being read on a cytometer.

 

A guide to the fixatives used in this experiment:

 

 

Human PBMCs were stained with anti-human CD4 (clone SK3) Spark NIR™ 685 and fixed using the respective protocols for each buffer set. Fresh samples were fixed and read on a cytometer immediately following fixation. Overnight samples were fixed and then stored in Cyto-Last™ Buffer (overnight) before reading.

Spark YG™ 570

 

Ideal for building multicolor microscopy panels, Spark YG™ 570 provides a bright and photostable signal. With an excitation max of 555 nm and an emission max of 570 nm, it can be imaged using the filter sets commonly used for Alexa Fluor® 555, Cy3, or TRITC in either widefield or confocal microscopy. While Spark YG™ 570 can be detected on a spectral cytometer, it is difficult to accurately unmix from PE due to a high degree of spectral similarity.

 

 

Spark YG 570 spectra

Normalized excitation and emission spectra of Spark YG™ 570 obtained from a spectrophotometer. To compare Spark YG™ 570 with other fluorophores, use our Fluorescence Spectra Analyzer tool.

 

Build Multicolor Microscopy Panels with Spark YG™ 570

 

Depending on the filter sets and light source of your microscope, Spark YG™ 570 can be used in combination with a variety of other fluorophores including DAPI, Alexa Fluor® 488, Alexa Fluor® 647, and Alexa Fluor® 750.

 

 

Immunohistochemistry on frozen mouse spleen tissue. Tissue on the left was stained with anti-CD3 Spark YG™ 570 (red), anti-B220 Alexa Fluor® 647 (green) and DAPI (blue). Tissue on the right was stained with anti-CD45 Spark YG™ 570 (red), anti-CD3 Alexa Fluor® 647 (green), and anti-B220 Alexa Fluor® 647 (blue).

 

Spark YG™ 570 Provides Similar Brightness to Alexa Fluor® 555

 

Spark YG 570 compared to Alexa Fluor 555

Rat (left and middle) or mouse (right) brain sections were stained with the indicated antibody conjugated to either Spark YG™ 570 or Alexa Fluor® 555. Across clones, tissues showed a similar brightness and staining pattern between the two antibodies.

 

Spark YG™ 570 Demonstrates Minimal Bleed-through into Nearby Channels

 

Spark YG 570 Bleedthrough

Human substantia nigra tissue was stained with anti-Tyrosine Hydrolase Spark YG™ 570 and imaged under a 40x objective. No bleed-through was observed in the 488 nm and 647 nm channels, even when using a five-fold longer exposure time.

 

Photostability of Spark YG™ 570

 

To test the photostability of Spark YG™ 570, we stained tissue sections with a purified antibody, followed by incubation with either an Alexa Fluor® 555 or Spark YG™ 570 conjugated secondary antibody. After staining, images were captured by exposing tissue sections to light at the specified time points.

 

Photostability Spark YG 570

FFPE human tonsil tissues were stained with purified anti-CD8a (Clone C8/144B) followed by incubation with Alexa Fluor® 555 or Spark YG™ 570 goat-anti-mouse IgG (Poly4053). Images were captured at the indicated time points and mean fluorescence intensity, expressed as Arbitrary Units (AU) was measured using ImageJ.

Spark Violet™ 538

 

Spark Violet™ 538 slots in as an additional violet laser fluor option for multicolor flow cytometry users. This dye is optimally excited by the 405 nm violet laser and emits maximally at 538 nm, placing its peak emission between Brilliant Violet 510™ and Brilliant Violet 570™. When used on a spectral cytometer, Spark Violet™ 538 can be successfully unmixed from neighboring dyes, such as BV510™, BV570™, and BV605™. While it is brighter than Pacific Orange™, Spark Violet™ 538 is relatively dim and should be paired with antigens expressed at high levels.

 

 

Excitation and Emission Spectra of Spark Violet™ 538

 

Spark Violet 538 Spectra Signature

 

Emission spectra (top) and normalized emission spectra (middle) of Spark Violet™ 538 run on a 5-laser Cytek™ Aurora Spectral Cytometer. To compare Spark Violet™ 538 with other fluorophores on a spectral cytometer, use our Aurora Spectral Analyzer tool.

 

Normalized excitation and emission spectra of Spark NIR™ 685 obtained from a spectrophotometer (bottom). To compare Spark Violet™ 538 with other fluorophores, use our Fluorescence Spectra Analyzer tool.

A Perfect Complement to Brilliant Violet™ Dyes

In the data below, Spark Violet™ 538 is utilized with several neighboring Brilliant Violet™ dyes, showcasing its ability to be spectrally unmixed from these dyes in a large, multicolor panel. 

 

Spark Violet 538 panel

 

Lysed human whole blood was stained with the indicated antibodies in the presence of Brilliant Stain Buffer. Samples were unmixed on a Cytek™ Aurora Cytometer using compensation beads and cells. All plots are gated on lymphocytes.

 

A New Alternative Pacific Orange™

Spark Violet™ 538 is spectrally similar to Pacific Orange™ with a peak emission in the V8 channel on the Cytek™ Aurora. Spark Violet™ 538 offers a brighter signal when compared to Pacific Orange™ in a side-by-side experiment.

 

Spark Violet 538 Titration

 

(Left) Human lysed whole blood was stained with anti-human CD4, clone SK3, conjugated to either Spark Violet™ 538 (solid purple) or Pacific Orange™ (clone S3.5, clear dashed) and then analyzed on a Cytek™ Aurora cytometer. Plots are gated on lymphocytes. (Right) Titration curve generated by staining human lysed whole blood with Spark Violet™ 538 conjugated to anti-CD4, clone SK3, antibody.  

 

Stability and Validation Testing

 

All BioLegend fluorophores undergo rigorous testing procedures to determine how light, heat, and fixation may affect the performance and ensure they will perform reliably. To compare the signal across different conditions and timepoints, we used the Stain Index (formula below) to measure the relative brightness of the antibody

Spark Violet 538 equation

 

Photostability Testing

 

The photostability of Spark Violet™ 538 was tested in two ways that mimic how an antibody may be exposed to light over the course of an experiment.

 

  1. Antibodies were stored in the dark or exposed to fluorescent lighting. Then, the antibodies were used to stain freshly harvested cell samples and analyzed immediately.
  2. Cells were stained with antibody that had been kept under recommended storage conditions. Prior to analysis, the stained cells were stored in the dark or exposed to fluorescent lighting.

Spark Violet 538

Anti-human CD4 Spark Violet™ 538 (clone SK3) or Pacific Orange™ (clone S3.5) antibodies were stored in a clear vial (dye only) and left exposed to light or protected in the dark, as indicated. Antibodies were stored for the indicated timepoints and then used to stain human lysed whole blood. “Dark” samples were only tested at the 24 hour time point as a control. Samples labeled "dye + cells" contain human lysed whole blood that was stained with the indicated antibody. Stained cells were then left in the light or protected, as indicated. 

 

Heat Stability

 

Spark Violet 538 Heat Stability

Anti-human CD4 antibodies, clone SK3 Spark Violet™ 538 or clone S3.5 Pacific Orange™, were kept at either 4°C or 37°C for seven weeks. The antibodies were then used to stain human lysed whole blood.

 

 

Fixative Stability

 

Spark Violet™ 538 is compatible with BioLegend buffers, but may be sensitive to alcohol-based buffers. The end user will need to test to ensure their antigen's signal is maintained post-fixation.

 

A guide to the fixatives used in this experiment:

 

 

Human PBMCs were stained with anti-human CD4 (clone SK3) conjugated to Spark Violet™ 538 and fixed using the respective protocols for each buffer set. For intracellular staining buffers, additional antibody stains for Granzyme A or FOXP3 were performed. For the alcohols, 90% methanol and 70% ethanol were used. Samples were then transferred into FluoroFix™ Buffer and read on a 5-laser Cytek™ Aurora Cytometer immediately.

Spark YG™ 581

 

Spark YG™ 581 joins the Spark Dyes as a novel fluorophore designed to be used on a 5-laser spectral cytometer. It is excited primarily by the 561 nm yellow/green laser and has a peak emission at 581 nm. Although it emits in the same channel as PE, Spark YG™ 581 has minimal cross-beam excitation with the 488 nm laser, making Spark YG™ 581 distinguishable from PE and allowing these highly overlapping fluorophores to be accurately unmixed. As such, Spark YG™ 581 is not recommended for 3-laser spectral cytometry. Spark YG™ 581 is recommended for an intermediate density antigen. 

 

 

 

Excitation and Emission Spectra of Spark YG™ 581

 

Spark YG 581 spectra signature

 

Emission spectra (top) and normalized emission spectra (middle) of Spark YG™ 581 run on a 5-laser Cytek™ Aurora Spectral Cytometer. To compare Spark YG™ 581 with other fluorophores on a spectral cytometer, use our Aurora Spectral Analyzer tool.

 

Normalized excitation and emission spectra (bottom) of Spark YG™ 581 obtained from a spectrophotometer. To compare Spark YG™ 581 with other fluorophores, use our Fluorescence Spectra Analyzer tool.

 

Unmix Spark YG™ 581 from PE and Yellow/Green Laser Options

 

Panel A demonstrates how Spark YG™ 581 performs in a multicolor panel with fluorophores that share significant spectra overlap with it. Panel B is shown as a reference panel using pre-optimized fluorophores to ensure that staining patterns are similar.

 

Spark YG 581 Panel

 

 

Marker

Panel A Fluorophore

Panel B Fluorophore

CD4

Spark YG™ 581

Spark YG™ 581

CD8

FITC

FITC

CD19

PE/Dazzle™ 594

PE/Cyanine7

CD56

Brilliant Violet™ 605

Brilliant Violet™ 421

CD335

PE

Alexa Fluor® 647

 

Human lysed whole blood was stained with the indicated antibodies and unmixed on a 5-laser Cytek™ Aurora Cytometer using cells. All plots are gated on lymphocytes.

 

 

Stability and Validation Testing

 

All BioLegend fluorophores undergo rigorous testing procedures to determine how light, heat, and fixation may affect the performance and ensure they will perform reliably. To compare the signal across different conditions and timepoints, we used the Stain Index (formula below) to measure the relative brightness of the antibody. 

Spark 550

Titration of Spark YG™ 581

Spark YG 581 Titration

Titration curve generated by staining human lysed whole blood with Spark YG™ 581 conjugated anti-human CD4 (SK3) antibody.

 

Photostability Testing

 

The photostability of Spark YG™ 581 was tested in two ways that mimic how an antibody may be exposed to light over the course of an experiment.

 

  1. Antibodies were stored in the dark or exposed to fluorescent lighting. Then, the antibodies were used to stain freshly harvested cell samples and analyzed immediately.
  2. Cells were stained with antibody that had been kept under recommended storage conditions. Prior to analysis, the stained cells were stored in the dark or exposed to fluorescent lighting.

 

Spark YG 581 Photostability

Anti-human CD4 (clone SK3) Spark YG™ 581 was stored in a clear vial (Ab only) and left exposed to light or protected in the dark, as indicated. Antibodies were stored for the indicated timepoints and then used to stain human lysed whole blood. Samples labeled Ab + cells contain human lysed whole blood that was stained with anti-human CD4 Spark YG™ 581. Stained cells were then left in the light or protected, as indicated.

 

Heat Stability

 

Spark YG 581 Heat Stability

Anti-human CD4 (clone SK3) Spark YG™ 581 was aliquoted and incubated at the indicated temperatures over the course of 28 days. The antibodies were then used to stain human lysed whole blood from a single donor.

 

Fixative Stability

 

Spark YG™ 581 is compatible with all BioLegend fixation buffers. For each buffer set, fresh fixed samples were tested immediately following staining or stored overnight in Cyto-Last™ Buffer before being read on a cytometer.

 

A guide to the fixatives used in this experiment:

 

 

Spark YG 581 Fixative

Human PBMCs were stained with anti-human CD4 (clone SK3) conjugated to Spark YG™ 581 and fixed using the respective protocols for each buffer set. Fresh samples were fixed and read on a 5-laser Cytek™ Aurora Cytometer immediately. Overnight samples were fixed and stored overnight in Cyto-Last™ Buffer before reading.

Spark YG™ 593

 

Spark YG™ 581 joins the Spark Dyes as a novel fluorophore designed to be used on a 5-laser spectral cytometer. It is excited primarily by the 561 nm yellow/green laser and has a peak emission at 581 nm. Although it emits in the same channel as PE, Spark YG™ 581 has minimal cross-beam excitation with the 488 nm laser, making Spark YG™ 581 distinguishable from PE and allowing these highly overlapping fluorophores to be accurately unmixed. As such, Spark YG™ 581 is not recommended for 3-laser spectral cytometry. Spark YG™ 581 is recommended for an intermediate density antigen. 

 

 

 

Excitation and Emission Spectra of Spark YG™ 581

 

Spark YG 581 spectra signature

 

Emission spectra (top) and normalized emission spectra (middle) of Spark YG™ 581 run on a 5-laser Cytek™ Aurora Spectral Cytometer. To compare Spark YG™ 581 with other fluorophores on a spectral cytometer, use our Aurora Spectral Analyzer tool.

 

Normalized excitation and emission spectra (bottom) of Spark YG™ 581 obtained from a spectrophotometer. To compare Spark YG™ 581 with other fluorophores, use our Fluorescence Spectra Analyzer tool.

 

Unmix Spark YG™ 581 from PE and Yellow/Green Laser Options

 

Panel A demonstrates how Spark YG™ 581 performs in a multicolor panel with fluorophores that share significant spectra overlap with it. Panel B is shown as a reference panel using pre-optimized fluorophores to ensure that staining patterns are similar.

 

Spark YG 581 Panel

 

 

Marker

Panel A Fluorophore

Panel B Fluorophore

CD4

Spark YG™ 581

Spark YG™ 581

CD8

FITC

FITC

CD19

PE/Dazzle™ 594

PE/Cyanine7

CD56

Brilliant Violet™ 605

Brilliant Violet™ 421

CD335

PE

Alexa Fluor® 647

 

Human lysed whole blood was stained with the indicated antibodies and unmixed on a 5-laser Cytek™ Aurora Cytometer using cells. All plots are gated on lymphocytes.

 

 

Stability and Validation Testing

 

All BioLegend fluorophores undergo rigorous testing procedures to determine how light, heat, and fixation may affect the performance and ensure they will perform reliably. To compare the signal across different conditions and timepoints, we used the Stain Index (formula below) to measure the relative brightness of the antibody. 

Spark 550

Titration of Spark YG™ 581

Spark YG 581 Titration

Titration curve generated by staining human lysed whole blood with Spark YG™ 581 conjugated anti-human CD4 (SK3) antibody.

 

Photostability Testing

 

The photostability of Spark YG™ 581 was tested in two ways that mimic how an antibody may be exposed to light over the course of an experiment.

 

  1. Antibodies were stored in the dark or exposed to fluorescent lighting. Then, the antibodies were used to stain freshly harvested cell samples and analyzed immediately.
  2. Cells were stained with antibody that had been kept under recommended storage conditions. Prior to analysis, the stained cells were stored in the dark or exposed to fluorescent lighting.

 

Spark YG 581 Photostability

Anti-human CD4 (clone SK3) Spark YG™ 581 was stored in a clear vial (Ab only) and left exposed to light or protected in the dark, as indicated. Antibodies were stored for the indicated timepoints and then used to stain human lysed whole blood. Samples labeled Ab + cells contain human lysed whole blood that was stained with anti-human CD4 Spark YG™ 581. Stained cells were then left in the light or protected, as indicated.

 

Heat Stability

 

Spark YG 581 Heat Stability

Anti-human CD4 (clone SK3) Spark YG™ 581 was aliquoted and incubated at the indicated temperatures over the course of 28 days. The antibodies were then used to stain human lysed whole blood from a single donor.

 

Fixative Stability

 

Spark YG™ 581 is compatible with all BioLegend fixation buffers. For each buffer set, fresh fixed samples were tested immediately following staining or stored overnight in Cyto-Last™ Buffer before being read on a cytometer.

 

A guide to the fixatives used in this experiment:

 

 

Spark YG 581 Fixative

Human PBMCs were stained with anti-human CD4 (clone SK3) conjugated to Spark YG™ 581 and fixed using the respective protocols for each buffer set. Fresh samples were fixed and read on a 5-laser Cytek™ Aurora Cytometer immediately. Overnight samples were fixed and stored overnight in Cyto-Last™ Buffer before reading.

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