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](/Files/Images/BioLegend/spark-dyes/equation.PNG)
Titration of Spark Blue™ 550
![](/Files/Images/BioLegend/spark-dyes/SpkBlue550_Titration.JPG)
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.
- 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.
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.
![](/Files/Images/BioLegend/spark-dyes/SpkBlue550_photo.JPG)
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
![](/Files/Images/BioLegend/spark-dyes/SpkBlue550_heat.JPG)
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](/Files/Images/BioLegend/spark-dyes/SparkBlue550_fix.JPG)
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.
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