Spark NIR 685

Excitation and Emission Spectra of Spark NIR™ 685

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.

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.

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.

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

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.

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:

• 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.

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.