The Language of Flow Cytometry and Experimental Setup
The language of flow cytometry inadvertently gives the impression that this technique is difficult. However, it is not; there is just a lot of jargon to learn like in any other field. In this chapter, we will define basic terminology that will enable end users to understand concepts important for experimental setup and how to interpret flow cytometry data. In particular, we will discuss FSC and SSC in detail with emphasis on gating strategies and how dead cell exclusion can be used to clean up your data. We will also outline how to interpret flow data on commonly used data plots including histograms, dot plots, and density plots. We will describe the differences between biexponential versus logarithmic scaling. We will define what CS&T is, why it is necessary, and how to apply all these concepts in basic experimental setup. Finally, we will conclude with the fundamental principles of compensation.
KeywordsFSC SSC MFI Geometric mean MFI Median MFI Mean MFI Gating Polygon gate Hierarchy Quadrants Histograms Dot plots Density plots Doublet exclusion Fluorescence Biexponential scaling Logarithmic scaling CS&T iTregs Voltages FACSDIVA™ software FlowJo® Compensation Compensation beads Spectral overlap Bandpass filters Spectrum viewers
- A405 =
Alexa Fluor® 405
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- 1.Mean, Median, Mode. FlowJo templates part 2. 2007. http://flowjo.typepad.com/the_daily_dongle/2007/10/mean-median-mod.html.