StarBright UltraViolet 575

Excitation and Emission Spectra of StarBright UltraViolet 575

Emission spectra (top) and normalized emission spectra (middle) of StarBright UV575 run on a 5-laser Cytek™ Aurora Spectral Cytometer. To compare StarBright UV575 with other fluorophores on a spectral cytometer, use our Aurora Spectral Analyzer tool.

Normalized excitation and emission spectra (bottom) of StarBright UV575 obtained from a spectrophotometer. To compare StarBright UV575 with other fluorophores, use our Fluorescence Spectra Analyzer tool.

Go Beyond BD Horizon™ BUV563

Human peripheral blood lymphocytes were stained with anti-human CD3 (clone UCHT1) Spark Violet™ 423 and anti-human CD4 (clone SK3) conjugated to StarBright UV575 and compared BD Horizon™ BUV563. Samples were acquired on a 5-laser Cytek® Aurora.

Multicolor Staining With StarBright UltraViolet 575

Panel A demonstrates how StarBright UV575 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.

Human PBMCs were stained with the indicated antibodies and analyzed on a spectral flow cytometer. 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 StarBright UV575 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 StarBright UV575-conjugated antibodies, anti-human CD4 (clone SK3) StarBright UV575 was exposed to light or protected in the dark for the indicated times (AB Only). The samples were then used to stain human PBMCs. To measure the photostability of StarBright UV575 stained cells, human PBMCs were stained with anti-human CD4 (clone SK3) StarBright UV575 (+ Cells). Stained cells were exposed to light or kept in the dark as indicated and the staining index of lymphocytes was calculated.

Heat Stability

Anti-human CD4 (clone SK3) StarBright UV575 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 and the staining index was calculated.

Fixative Stability

A guide to the fixatives used in this experiment:

• FluoroFix™ is a gentler fixation PFA-based buffer.
• Cyto-Fast™ Fix/Perm Buffer Set is a specialized buffer for cytokine detection and offers improved staining over the Intracellular Staining Permeabilization Wash Buffer.
• 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 phospho protein targets.
• Cyto-Last™ Buffer is formulated for long-term storage of samples while preserving signal over time.
• Ethanol or methanol 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 StarBright UV575 and fixed using the respective protocols for each buffer set. Fresh samples were fixed and read on a 5-laser Cytek™ Aurora spectral cytometer immediately. Overnight samples were fixed and stored in Cell Staining Buffer overnight before acquisition. Data shown was gated on lymphocytes.

StarBright UltraViolet 740

Excitation and Emission Spectra of StarBright UltraViolet 740

Emission spectra (top) and normalized emission spectra (middle) of StarBright UV740 run on a 5-laser Cytek™ Aurora Spectral Cytometer. To compare StarBright UV740 with other fluorophores on a spectral cytometer, use our Aurora Spectral Analyzer tool.

Normalized excitation and emission spectra (bottom) of StarBright UV740 obtained from a spectrophotometer. To compare StarBright UV740 with other fluorophores, use our Fluorescence Spectra Analyzer tool.

Go Beyond BD Horizon™ BUV737

Human peripheral blood lymphocytes were stained with anti-human CD3 (clone UCHT1) Spark Violet™ 423 and anti-human CD4 (clone SK3) conjugated to StarBright UV740 and compared against BD Horizon™ BUV737. Samples were acquired on a 5-laser Cytek® Aurora.

Multicolor Staining With StarBright UltraViolet 740

Panel A demonstrates how StarBright UV740 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.

Human peripheral blood cells were stained with the indicated antibodies and analyzed on a spectral flow cytometer. 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 StarBright UV740 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 StarBright UV740-conjugated antibodies, anti-human CD4 (clone SK3) StarBright UV740 was exposed to light or protected in the dark for the indicated times (AB Only). The samples were then used to stain human PBMCs. To measure the photostability of StarBright UV740 stained cells, human PBMCs were stained with anti-human CD4 (clone SK3) StarBright UV740 (+ Cells). Stained cells were exposed to light or kept in the dark as indicated and the staining index of lymphocytes was calculated.

Heat Stability

Anti-human CD4 (clone SK3) StarBright UV740 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, and the staining index of lymphocytes was calculated.

Fixative Stability

A guide to the fixatives used in this experiment:

• FluoroFix™ is a gentler fixation PFA-based buffer.
• Cyto-Fast™ Fix/Perm Buffer Set is a specialized buffer for cytokine detection and offers improved staining over the Intracellular Staining Permeabilization Wash Buffer.
• 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 phospho protein targets.
• Cyto-Last™ Buffer is formulated for long-term storage of samples while preserving signal over time.
• Ethanol or methanol 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 StarBright UV740 and fixed using the respective protocols for each buffer set. Fresh samples were fixed and read on a 5-laser Cytek™ Aurora spectral cytometer immediately. Overnight samples were fixed and stored in Cell Staining Buffer overnight before acquisition. Data shown was gated on lymphocytes.

StarBright UltraViolet 795

StarBright™ UltraViolet 795 exhibits peak excitation/emission wavelengths at 340 nm and 792 nm respectively, most closely matching that of BD Horizon™ BUV805. On conventional cytometers, it can be detected with a 355 nm ultraviolet laser and a 820/60 filter set (or equivalent). With proper panel building, it can be used together in panels with StarBright UV575, StarBright UV740, and Spark PLUS UV395™. This extremely bright fluorophore is best suited for detecting antigens with low expression levels. In addition, StarBright UV795 can be cocktailed with other fluorophore conjugated antibodies without exhibiting inter-dye interactions. It is also stable towards exposure to heat or a variety of commonly used fixative solutions.

Excitation and Emission Spectra of StarBright UltraViolet 795

Emission spectra (top) and normalized emission spectra (middle) of StarBright UV795 run on a 5-laser Cytek™ Aurora Spectral Cytometer. To compare StarBright UV795 with other fluorophores on a spectral cytometer, use our Aurora Spectral Analyzer tool.

Normalized excitation and emission spectra (bottom) of StarBright UV795 obtained from a spectrophotometer. To compare StarBright UV795 with other fluorophores, use our Fluorescence Spectra Analyzer tool.

Go Beyond BD Horizon™ BUV805

Human peripheral blood lymphocytes were stained with anti-human CD3 (clone UCHT1) Spark Violet™ 423 and anti-human CD4 (clone SK3) conjugated to StarBright UV795 and compared against BD Horizon™ BUV805. Samples were acquired on a 5-laser Cytek® Aurora.

Multicolor Staining With StarBright UltraViolet 795

Panel A demonstrates how StarBright UV795 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 peripheral blood cells were stained with the indicated antibodies and analyzed on a spectral flow cytometer. 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 StarBright UV795 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 StarBright UV795-conjugated antibodies, anti-human CD4 (clone SK3) StarBright UV795 conjugates were exposed to light or protected in the dark (Ab Only). The samples were then used to stain human PBMCs. To measure the photostability of StarBright UV795 stained cells, human PBMCs were stained with anti-human CD4 (clone SK3) StarBright UV795 (+ Cells). Stained cells were exposted to light or kept in the dark as indicated, and the staining index of lymphocytes was calculated. 

Heat Stability

Anti-human CD4 (clone SK3) StarBright UV795 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, and the staining index of lymphocytes was calculated.

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 or methanol 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 StarBright UV795 and fixed using the respective protocols for each buffer set. Fresh samples were fixed and read on a 5-laser Cytek™ Aurora spectral cytometer immediately. Overnight samples were fixed and stored in Cell Staining Buffer overnight before acquisition. Data shown was gated on lymphocytes.