Game 3: Photostability
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Fluorescent dyes can undergo photobleaching (dimming) when exposed to light. This can cause a decrease in resolution. Additionally, some dyes may undergo spectral changes when exposed to light. In this Fluorochrome Faceoff, we evaluate the impact of light on fluorochromes that are primarily excited by the yellow-green laser with peak emission around 700 nm.
Human whole blood was stained with BD Horizon™ RY703 Mouse Anti-Human CD4 SK3 (Cat. No. 571427), Bio-Rad StarBright™ Yellow 720 Mouse Anti-Human CD4 RPA-T4 or Thermo Fisher NovaFluor™ Y700 Mouse Anti-Human CD4 SK3 Reagent. Erythrocytes were lysed with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899). After staining was completed, cells were either kept in the dark at room temperature (purple histogram) or exposed to 200 lux of LED light at room temperature for 2 (blue histogram) or 4 hours (green histogram). Histograms were derived from gated events based on light scattering characteristics for lymphocytes. Flow cytometry and data analysis were performed using a BD FACSymphony™ A5 SE Cell Analyzer System and FlowJo™ Software.
Note that in the histograms above, both MFI loss and an increase in background with light exposure can be observed. Both changes result in lower stain index and resolution.
Winner’s Circle
Exposure to light can cause changes in a fluorochrome's emission profile, measured here as changes in spillover into other detectors. In an experimental setting, this can impact researchers as differences in spectral profile between reference controls and samples can lead to compensation or unmixing errors and data artifacts. This example shows changes in spillover in several detectors after 2 hours of light exposure, with SBY720 being the fluorochrome impacted the most. SBY720 also exhibited the greatest loss in resolution after light exposure.
Our winner for this Fluorochrome Faceoff is RY703 because this fluorochrome exhibited the least amount of spillover change and MFI loss across the dyes tested in this game.
PRO TIP: Keep your tubes protected from light during sample acquisition to prevent changes in spectral profiles over time.
Runner-Up:
NFY700
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Disclaimer: Results and conclusions shown throughout the Fluorochrome Faceoff are based on experiments performed under the conditions described. Users should evaluate reagents with their specific protocols as results may vary with different experimental conditions.
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