Game 2: Spillover
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Fluorescence spillover is the fluorescent signal measured in adjacent, residual and cross-laser detectors. Spectral overlap between fluorochromes can be managed through compensation or spectral unmixing to prevent data artifacts. However, even with compensation and unmixing, spectral overlap of two fluorochromes in a panel can lead to compromised resolution through spillover spreading and unmixing error, making spillover an important consideration in panel design.
Spillover here is evaluated and ranked based on the analysis of a given fluorochrome’s full emission profile across five lasers.
Fluorochromes with less cross-laser excitation are cleaner and will have lower impact on other fluorochromes. We evaluated several fluorochromes primarily excited by the blue laser that have peak emission around 700 nm:
BD Horizon RealBlue™ 705 (RB705)
BD Horizon Brilliant™ Blue 700 (BB700)
Bio-Rad StarBright™ Blue 700 (SBB700)
BD Pharmingen™ PerCP-Cy5.5
Thermo Fisher PerCP-eFluor™ 710
Thermo Fisher NovaFluor™ Blue 690 (NFB690)
Freshly isolated human PBMCs were stained with either BD Horizon™ RB705 Mouse Anti-Human CD4 SK3 (Cat. No. 570221), BD Pharmingen™ PerCP-Cy5.5 Mouse Anti-Human CD4 SK3 (Cat. No. 566923), BD Horizon™ BB700 Mouse Anti-Human CD4 SK3 (Cat. No. 566392), Thermo Fisher PerCP-eFluor™ 710 Mouse Anti-Human CD4 SK3, Bio-Rad StarBright™ Blue 700 Mouse Anti-Human CD4 RPA-T4, or Thermo Fisher NovaFluor™ Blue 690 Anti-Human CD4 SK3 Reagent. Spectral profiles are generated from the positive population with the negative subtracted and are normalized to peak detector. Spillover percentages are dependent on the configuration of an instrument. The rankings were based on analysis on three instruments (BD FACSDiscover™ S8 Cell Sorter, BD FACSymphony™ A5 SE Cell Analyzer and Cytek Aurora). For the data shown, flow cytometry and data analysis were performed using a BD FACSDiscover™ S8 Cell Sorter and FlowJo™ Software.
Winner’s Circle
Using normalized emission profiles, winners were determined based on the number of lasers with an additional peak of signal greater than 15% of the main peak signal. RB705 and NFB690 had the least amount of emission into other channels and are therefore declared the winners!
PRO TIP: When assigning a fluorochrome to a marker, it is important to consider how spillover spreading may affect resolution of other markers in your panel.
Runner-Up:
BB700
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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.
BD flow cytometers are Class 1 Laser Products. For Research Use Only. Not for use in diagnostic or therapeutic procedures.
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