Spark Dyes

Spark PLUS B574™

Spark PLUS B574™ is excited by the 488 nm blue laser and, while suitable for conventional instruments with a 574/40 filter, demonstrates maximum brightness on spectral cytometers. Spark PLUS B574™ is ideal for combining with other blue laser-excited dyes in large spectral panels, as demonstrated by its ability to be unmixed from dyes like Spark PLUS B550™, FITC, and PerCP/Cyanine5.5. It can also be unmixed from PE when using a spectral instrument with both blue and yellow-green lasers.

Excitation and Emission Spectra of Spark PLUS B574™

Emission spectra (top) and normalized emission spectra (middle) of Spark PLUS B574™ run on a 5-laser Cytek® Aurora Spectral Cytometer. To compare Spark PLUS B574™ with other fluorophores on a spectral cytometer, use our Aurora Spectral Analyzer tool.

Normalized excitation and emission spectra (bottom) of Spark PLUS B574™ obtained from a spectrophotometer. To compare Spark PLUS B574™ with other fluorophores, use our Fluorescence Spectra Analyzer tool.

Multicolor Staining with Spark PLUS B574™

Panel A demonstrates how Spark PLUS B574™ performs in a multicolor panel with fluorophores that may exhibit spectral overlap with it. Panel B is shown as a reference panel using pre-optimized fluorophores to ensure that staining patterns are similar.

Human lysed whole blood cells were stained with the indicated antibodies and analyzed on 5-laser Cytek® Aurora. 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. 

Photostability Testing

The photostability of Spark PLUS B574™ 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.

To assess the photostability of Spark PLUS B574™-conjugated antibodies, anti-human CD4 (clone SK3) Spark PLUS B574™ was exposed to light (blue) or protected in the dark (red) for the indicated time points. The samples were then used to stain human PBMCs. To measure the photostability of Spark PLUS B574™ stained cells, human PBMCs were stained with anti-human CD4 (clone SK3) Spark PLUS B574™. Stained cells were then left in the light (green) or protected in the dark (purple) as indicated. Cells in the lymphocyte gate were used for analysis.

Heat Stability

Anti-human CD4 (clone SK3) Spark PLUS B574™ was aliquoted and incubated at the indicated temperatures over the course of 28 days, and then analyzed on a 5-laser Cytek® Aurora cytometer. The antibodies were then used to stain human lysed whole blood from a single donor. Cells in the lymphocyte gate were used for analysis.

Fixative Stability

A guide to the fixatives used in this experiment:

• FluoroFix™ is a gentler PFA-based fixation buffer.
• Cyto-Fast™ Fix/Perm Buffer Set is a specialized buffer for cytokine detection.
• True-Nuclear™ Transcription Factor Buffer Set is designed for optimal staining of intranuclear targets.
• True-Phos™ Perm Buffer is an alcohol-based buffer ideal for staining of phosphorylated protein targets.
• Cyto-Last™ Buffer is formulated for long-term storage of samples while preserving signal over time.
• Ethanol can be a component of fixation and permeabilization buffers. Some fluors, particularly protein-based ones, can be more sensitive to alcohol-based treatments than others.

Human PBMCs were stained with anti-human CD4 (clone SK3) conjugated to Spark PLUS B574™ and fixed using the respective protocols for each buffer set. Fresh samples were fixed and read on a Cytek® cytometer immediately. Overnight samples were fixed and stored in Cell Staining Buffer overnight before acquisition. Data shown was gated on lymphocytes.

Spark PLUS B488™

Spark PLUS B488™ is excited by the blue (488 nm) laser and is suitable for use on both conventional and spectral instruments. It offers improved brightness compared to traditional fluorophores like FITC and Alexa Fluor® 488.  Spark PLUS B488™ is ideal for combining with other dyes in large spectral panels, as demonstrated by its ability to be unmixed from dyes like Spark PLUS B550™, Spark PLUS B574™, and PE.

Excitation and Emission Spectra of Spark PLUS B488™

Emission spectra (top) and normalized emission spectra (middle) of Spark PLUS B488™ run on a 5-laser Cytek® Aurora Spectral Cytometer. To compare Spark PLUS B488™ with other fluorophores on a spectral cytometer, use our Aurora Spectral Analyzer tool.

Normalized excitation and emission spectra (bottom) of Spark PLUS B488™ obtained from a spectrophotometer. To compare Spark PLUS B488™ with other fluorophores, use our Fluorescence Spectra Analyzer tool.

Multicolor Staining with Spark PLUS B488™

Panel A demonstrates how Spark PLUS B488™ performs in a multicolor panel with fluorophores that may exhibit spectral overlap with it. Panel B is shown as a reference panel using pre-optimized fluorophores to ensure that staining patterns are similar.

Human lysed whole blood cells were stained with the indicated antibodies and analyzed on 5-laser Cytek® Aurora. 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. 

Photostability Testing

The photostability of Spark PLUS B488™ 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.

To assess the photostability of Spark PLUS B488™-conjugated antibodies, anti-human CD4 (clone SK3) Spark PLUS B488™ was exposed to light (blue) or protected in the dark (red) for the indicated time points. The samples were then used to stain human PBMCs. To measure the photostability of Spark PLUS B488™ stained cells, human PBMCs were stained with anti-human CD4 (clone SK3) Spark PLUS B488™. Stained cells were then left in the light (green) or protected in the dark (purple) as indicated. Cells in the lymphocyte gate were used for analysis.

Heat Stability

Anti-human CD4 (clone SK3) Spark PLUS B488™ was aliquoted and incubated at the indicated temperatures over the course of 28 days, and then analyzed on a 5-laser Cytek® Aurora cytometer. The antibodies were then used to stain human lysed whole blood from a single donor. Cells in the lymphocyte gate were used for analysis.

Fixative Stability

A guide to the fixatives used in this experiment:

• FluoroFix™ is a gentler PFA-based fixation buffer.
• Cyto-Fast™ Fix/Perm Buffer Set is a specialized buffer for cytokine detection.
• True-Nuclear™ Transcription Factor Buffer Set is designed for optimal staining of intranuclear targets.
• True-Phos™ Perm Buffer is an alcohol-based buffer ideal for staining of phosphorylated protein targets.
• Cyto-Last™ Buffer is formulated for long-term storage of samples while preserving signal over time.
• Ethanol can be a component of fixation and permeabilization buffers. Some fluors, particularly protein-based ones, can be more sensitive to alcohol-based treatments than others.

Human PBMCs were stained with anti-human CD4 (clone SK3) conjugated to Spark PLUS B488™ and fixed using the respective protocols for each buffer set. Fresh samples were fixed and read on a Cytek® cytometer immediately. Overnight samples were fixed and stored in Cell Staining Buffer overnight before acquisition. Data shown was gated on lymphocytes.

Spark PLUS YG581™

Spark PLUS YG581™ is excited by the yellow/green (561 nm) laser and is suitable for use on both conventional and spectral instruments. Spark PLUS YG581™ emits in the same channel as PE, but has minimal cross-beam excitation with the 488 nm laser, making it distinguishable from PE and allowing these highly overlapping fluorophores to be accurately unmixed on a spectral cytometer. Spark PLUS YG581™ is ideal for combining with other dyes in large spectral panels, as demonstrated by its ability to be unmixed from dyes like Brilliant Violet 605™, PE/Dazzle™ 594, FITC, and PE.

Excitation and Emission Spectra of Spark PLUS YG581™

Emission spectra (top) and normalized emission spectra (middle) of Spark PLUS YG581™ run on a 5-laser Cytek® Aurora Spectral Cytometer. To compare Spark PLUS YG581™ with other fluorophores on a spectral cytometer, use our Aurora Spectral Analyzer tool.

Normalized excitation and emission spectra (bottom) of Spark PLUS YG581™ obtained from a spectrophotometer. To compare Spark PLUS YG581™ with other fluorophores, use our Fluorescence Spectra Analyzer tool.

Multicolor Staining with Spark PLUS YG581™

Panel A demonstrates how Spark PLUS YG581™ performs in a multicolor panel with fluorophores that may exhibit spectral overlap with it. Panel B is shown as a reference panel using pre-optimized fluorophores to ensure that staining patterns are similar.

Human lysed whole blood cells were stained with the indicated antibodies and analyzed on 5-laser Cytek® Aurora. 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. 

Photostability Testing

The photostability of Spark PLUS YG581™ 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.

To assess the photostability of Spark PLUS YG581™-conjugated antibodies, anti-human CD4 (clone SK3) Spark PLUS YG581™ was exposed to light (blue) or protected in the dark (red) for the indicated time points. The samples were then used to stain human PBMCs. To measure the photostability of Spark PLUS YG581™ stained cells, human PBMCs were stained with anti-human CD4 (clone SK3) Spark PLUS YG581™. Stained cells were then left in the light (green) or protected in the dark (purple) as indicated. Cells in the lymphocyte gate were used for analysis.

Heat Stability

Anti-human CD4 (clone SK3) Spark PLUS YG581™ was aliquoted and incubated at the indicated temperatures over the course of 28 days, and then analyzed on a 5-laser Cytek® Aurora cytometer. The antibodies were then used to stain human lysed whole blood from a single donor. Cells in the lymphocyte gate were used for analysis.

Fixative Stability

A guide to the fixatives used in this experiment:

• FluoroFix™ is a gentler PFA-based fixation buffer.
• Cyto-Fast™ Fix/Perm Buffer Set is a specialized buffer for cytokine detection.
• True-Nuclear™ Transcription Factor Buffer Set is designed for optimal staining of intranuclear targets.
• True-Phos™ Perm Buffer is an alcohol-based buffer ideal for staining of phosphorylated protein targets.
• Cyto-Last™ Buffer is formulated for long-term storage of samples while preserving signal over time.
• Ethanol can be a component of fixation and permeabilization buffers. Some fluors, particularly protein-based ones, can be more sensitive to alcohol-based treatments than others.

Human PBMCs were stained with anti-human CD4 (clone SK3) conjugated to Spark PLUS YG581™ and fixed using the respective protocols for each buffer set. Fresh samples were fixed and read on a Cytek® cytometer immediately. Overnight samples were fixed and stored in Cell Staining Buffer overnight before acquisition. Data shown was gated on lymphocytes.

Spark PLUS UV395™

Spark PLUS UV395™ is a bright UV laser-excited dye that can be used on both conventional and spectral flow cytometers. It exhibits peak excitation/emission wavelengths at 355 nm and 385 nm respectively, most closely matching that of BD Horizon™ BUV395. On conventional cytometers, it can be detected with a 355 nm laser and 379/28 filter set (or equivalent). With its intense brightness, Spark PLUS UV395™ is a versatile option for detecting antigens at most expression levels and is stable towards exposure to heat or a variety of commonly used fixative solutions. Spark PLUS UV395™ is the first in the family of Spark PLUS™ Dyes, all formulated to provide brighter signals and superior performance.

Excitation and Emission Spectra of Spark PLUS UV395™

Emission spectra (top) and normalized emission spectra (middle) of Spark PLUS UV395™ run on a 5-laser Cytek™ Aurora Spectral Cytometer. To compare Spark PLUS UV395™ with other fluorophores on a spectral cytometer, use our Aurora Spectral Analyzer tool.

Normalized excitation and emission spectra (bottom) of Spark PLUS UV395™ obtained from a spectrophotometer. To compare Spark PLUS UV395™ with other fluorophores, use our Fluorescence Spectra Analyzer tool.

A New Alternative to BD Horizon™ BUV395

Human peripheral blood lymphocytes were stained with anti-human CD3 APC and anti-human CD4 (clone SK3) Spark PLUS UV395™ (left) or anti-human CD4 (clone SK3) BD Horizon™ BUV395 (right). Samples were acquired on a 5-laser Cytek® Aurora.

Multicolor Staining With Spark PLUS UV395™

Panel A demonstrates how Spark PLUS UV395™ performs in a multicolor panel with fluorophores that may exhibit spectral overlap with it. Panel B is shown as a reference panel using pre-optimized fluorophores to ensure that staining patterns are similar.

Human PBMCs were stained with the indicated antibodies and analyzed on a conventional flow cytometer. All plots are gated on lymphocytes.

Unmix Spark PLUS UV395™ and Spark UV™ 387

Human PBMCs were stained with anti-human CD8 Spark UV 387 and anti-human CD4 (clone SK3) Spark PLUS UV395™ (left) or anti-human CD4 (clone SK3) APC (right). Samples were acquired on a 5-laser Cytek® Aurora cytometer.

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. 

Photostability Testing

The photostability of Spark PLUS UV395™ 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.

To assess the photostability of Spark PLUS UV395™-conjugated antibodies, anti-human CD4 (clone SK3) Spark PLUS UV395™ or BD Horizon™ BUV395 conjugates were exposed to light or protected in the dark (left). The samples were then used to stain human PBMCs and the fold change over the staining index at zero hours was calculated. To measure the photostability of Spark PLUS UV395™ stained cells, human PBMCs were stained with anti-human CD4 (clone SK3) Spark PLUS UV395™ or BD Horizon™ BUV395. The fold change in staining index of stained cells kept in the dark or exposed to light was calculated from the initial staining index at zero hours (right). Cells were gated on lymphocytes and data was acquired on a 5-laser Cytek® Aurora.

Heat Stability

Anti-human CD4 (clone SK3) Spark PLUS UV395™ was aliquoted and incubated at the indicated temperatures over the course of 28 days, and then analyzed on a 5-laser Cytek™ Aurora cytometer. The antibodies were then used to stain human lysed whole blood from a single donor.

Fixative Stability

A guide to the fixatives used in this experiment:

• FluoroFix™ is a gentler PFA-based fixation buffer.
• Cyto-Fast™ Fix/Perm Buffer Set is a specialized buffer for cytokine detection.
• True-Nuclear™ Transcription Factor Buffer Set is designed for optimal staining of intranuclear targets.
• True-Phos™ Perm Buffer is an alcohol-based buffer ideal for staining of phosphorylated protein targets.
• Cyto-Last™ Buffer is formulated for long-term storage of samples while preserving signal over time.
• Ethanol can be a component of fixation and permeabilization buffers. Some fluors, particularly protein-based ones, can be more sensitive to alcohol-based treatments than others.

Human PBMCs were stained with anti-human CD4 (clone SK3) conjugated to Spark PLUS UV395™ 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 in Cyto-Last™ Buffer overnight before reading.

Spark PLUS V475™

Spark PLUS V475™ is our newest fluorophore excited by the 405 nm violet laser, providing a bright, cleaner alternative to Brilliant™ Violet 480 with reduced spillover into BV510™. It can replace BV480™ on conventional instruments, while its unique spectral profile also allows clear unmixing from BV480™ on spectral instruments. Spark PLUS V475™ can also be unmixed from other violet laser options including BV421™, BV510™, Pacific Blue™, Spark Violet™ 423 and Spark Violet™ 500.

Excitation and Emission Spectra of Spark PLUS V475™

Emission spectra (top) and normalized emission spectra (middle) of Spark PLUS V475™ run on a 5-laser Cytek® Aurora Spectral Cytometer. To compare Spark PLUS V475™ with other fluorophores on a spectral cytometer, use our Aurora Spectral Analyzer tool.

Normalized excitation and emission spectra (bottom) of Spark PLUS V475™ obtained from a spectrophotometer. To compare Spark PLUS V475™ with other fluorophores, use our Fluorescence Spectra Analyzer tool.

Multicolor Staining with Spark PLUS V475™

Panel A demonstrates how Spark PLUS V475™ performs in a multicolor panel with fluorophores that may exhibit spectral overlap with it. Panel B is shown as a reference panel using pre-optimized fluorophores to ensure that staining patterns are similar.

Human lysed whole blood cells were stained with the indicated antibodies and analyzed on 5-laser Cytek® Aurora. All plots are gated on lymphocytes.

Unmixing Spark PLUS V475™ from BV480™

Panel A demonstrates how Spark PLUS V475™ performs when unmixed from BV480™ and other Brilliant Violet™ dyes on a spectral cytometer. Panel B is shown as a reference panel using pre-optimized fluorophores to ensure that staining patterns are similar.

Human lysed whole blood cells were stained with the indicated antibodies and analyzed on 5-laser Cytek® Aurora. 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. 

Photostability Testing

The photostability of Spark PLUS V475™ 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.

To assess the photostability of Spark PLUS V475™-conjugated antibodies, anti-human CD4 (clone SK3) Spark PLUS V475™ was exposed to light (blue) or protected in the dark (red) for the indicated time points. The samples were then used to stain human PBMCs. To measure the photostability of Spark PLUS V475™ stained cells, human PBMCs were stained with anti-human CD4 (clone SK3) Spark PLUS V475™. Stained cells were then left in the light (green) or protected in the dark (purple) as indicated. Cells in the lymphocyte gate were used for analysis.

Heat Stability

Anti-human CD4 (clone SK3) Spark PLUS V475™ was aliquoted and incubated at the indicated temperatures over the course of 28 days, and then analyzed on a 5-laser Cytek® Aurora cytometer. The antibodies were then used to stain human lysed whole blood from a single donor. Cells in the lymphocyte gate were used for analysis.

Fixative Stability

A guide to the fixatives used in this experiment:

• FluoroFix™ is a gentler PFA-based fixation buffer.
• Cyto-Fast™ Fix/Perm Buffer Set is a specialized buffer for cytokine detection.
• True-Nuclear™ Transcription Factor Buffer Set is designed for optimal staining of intranuclear targets.
• True-Phos™ Perm Buffer is an alcohol-based buffer ideal for staining of phosphorylated protein targets.
• Cyto-Last™ Buffer is formulated for long-term storage of samples while preserving signal over time.
• Ethanol can be a component of fixation and permeabilization buffers. Some fluors, particularly protein-based ones, can be more sensitive to alcohol-based treatments than others.

Human PBMCs were stained with anti-human CD4 (clone SK3) conjugated to Spark PLUS V475™ and fixed using the respective protocols for each buffer set. Fresh samples were fixed and read on a Cytek® cytometer immediately. Overnight samples were fixed and stored in Cell Staining Buffer overnight before acquisition. Data shown was gated on lymphocytes.

Spark PLUS B550™

Spark PLUS B550™ is formulated for brighter signal and superior stability, maximizing signal from the blue laser with minimal yellow/green cross-excitation and spillover. This fluorophore serves as an alternative to BD Horizon™ RealBlue545 and can be used on conventional and spectral flow cytometers, and is ideal for combining with PE and FITC on spectral instruments.

Excitation and Emission Spectra of Spark PLUS B550™

Emission spectra (top) and normalized emission spectra (middle) of Spark PLUS B550™ run on a 5-laser Cytek™ Aurora Spectral Cytometer. To compare Spark PLUS B550™ with other fluorophores on a spectral cytometer, use our Aurora Spectral Analyzer tool.

Normalized excitation and emission spectra (bottom) of Spark PLUS B550™ obtained from a spectrophotometer. To compare Spark PLUS B550™ with other fluorophores, use our Fluorescence Spectra Analyzer tool.

A New Alternative to BD Horizon™ RealBlue 545

Human peripheral blood lymphocytes were stained with anti-human CD19 (clone HIB19) Brilliant Violet 421™ and either CD4 (clone SK3) Spark PLUS B550™ (left) or CD4 (clone SK3) BD Horizon RealBlue™ 545 (right). Samples were acquired on a 5-laser Cytek® Aurora.

Multicolor Staining with Spark PLUS B550™

Panel A demonstrates how Spark PLUS B550™ performs in a multicolor panel with fluorophores that may exhibit spectral overlap with it. Panel B is shown as a reference panel using pre-optimized fluorophores to ensure that staining patterns are similar.

Human PBMCs were stained with the indicated antibodies and analyzed on 5-laser Cytek® Aurora. 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. 

Photostability Testing

The photostability of Spark PLUS B550™ 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.

To assess the photostability of Spark PLUS B550™-conjugated antibodies, anti-human CD4 (clone SK3) Spark PLUS B550™ was exposed to light or protected in the dark for the indicated time points. The samples were then used to stain human lysed whole blood. To measure the photostability of Spark PLUS B550™ stained cells, human lysed whole blood was stained with anti-human CD4 (clone SK3) Spark PLUS B550™. Stained cells were then left in the light or protected in the dark as indicated. Cells in the lymphocyte gate were used for analysis.

Heat Stability

Anti-human CD4 (clone SK3) Spark PLUS B550™ was aliquoted and incubated at the indicated temperatures over the course of 28 days, and then analyzed on a 5-laser Cytek™ Aurora cytometer. The antibodies were then used to stain human lysed whole blood from a single donor.

Fixative Stability

A guide to the fixatives used in this experiment:

• FluoroFix™ is a gentler PFA-based fixation buffer.
• Cyto-Fast™ Fix/Perm Buffer Set is a specialized buffer for cytokine detection.
• True-Nuclear™ Transcription Factor Buffer Set is designed for optimal staining of intranuclear targets.
• True-Phos™ Perm Buffer is an alcohol-based buffer ideal for staining of phosphorylated protein targets.
• Cyto-Last™ Buffer is formulated for long-term storage of samples while preserving signal over time.
• Ethanol can be a component of fixation and permeabilization buffers. Some fluors, particularly protein-based ones, can be more sensitive to alcohol-based treatments than others.

Human PBMCs were stained with anti-human CD4 (clone SK3) conjugated to Spark PLUS B550™ and fixed using the respective protocols for each buffer set. Fresh samples were fixed and read on a BD LSRFortessa™ cytometer immediately. Overnight samples were fixed and stored in Cyto-Last™ Buffer overnight before reading.