Flow Cytometry Gating: First Steps

Gating is the process by which specific populations of interest are isolated within a heterogenous sample. This allows the fluorescent properties of the populations of interest to be highlighted across all the available dot plots, increasing the specificity of the analysis.

Debris: Side Scatter and Forward Scatter

Dead cells and debris have a low forward scatter. This plot therefore allow debris to be easily excluded from analysis.

The SSc/FSc plot allow debris to be excluded reliably. Debris and dead cells have a low forward scatter, and can interfere with analysis if not excluded.

Doublets: Forward Scatter Height and Area

  • A doublet is a single event which consists of two independent particles.
  • They are classified as a single event as they pass through the interrogation point very close to each other – the instrument is incapable of distinguishing them.
  • Doublets can affect purity of sorting, population frequencies and staining patterns.
  • Higher sample flow rates can increase the proportion of doublets.
  • Doublets can be visualised using the FSC area (integral) vs FSC height (peak) plots.
Flow Cytometry: Doublet Exclusion
Doublet exclusion ensures that only single cells are included in the flow cytometry analysis. This is critical for reducing false positives in flow cytometry. Doublets can be identified on FSC-A vs FSC-H (area vs height) plots. They have a larger area compared to singlets, while maintaining a similar height.
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