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Spark PLUS UV™ 395

Spark PLUS UV™ 395 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 UV™ 395 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 UV™ 395 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 UV™ 395

Emission spectra (top) and normalized emission spectra (middle) of Spark PLUS UV™ 395 run on a 5-laser Cytek™ Aurora Spectral Cytometer. To compare Spark PLUS UV™ 395 with other fluorophores on a spectral cytometer, use our Aurora Spectral Analyzer tool.

Normalized excitation and emission spectra (bottom) of Spark PLUS UV™ 395 obtained from a spectrophotometer. To compare Spark PLUS UV™ 395 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 UV 395 (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 UV™ 395

Panel A demonstrates how Spark PLUS UV™ 395 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.

Marker	Panel A Fluorophore	Panel B Fluorophore
CD3	Spark Violet™ 538	FITC
CD4	Spark PLUS UV™ 395	Spark PLUS UV™ 395
CD8	FITC	APC
CD19	Brilliant Violet 570™	PE
CD25	Brilliant Violet 421™	Brilliant Violet 421™

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

Unmix Spark PLUS UV™ 395 and Spark UV™ 387

Human PBMCs were stained with anti-human CD8 Spark UV 387 and anti-human CD4 (clone SK3) Spark PLUS UV 395 (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 UV™ 395 was tested in two ways that mimic how an antibody may be exposed to light over the course of an experiment.

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.
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 UV™ 395-conjugated antibodies, anti-human CD4 (clone SK3) Spark PLUS UV™ 395 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 UV™ 395 stained cells, human PBMCs were stained with anti-human CD4 (clone SK3) Spark PLUS UV™ 395 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 UV™ 395 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 UV™ 395 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.