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Flow cytometry data analysis is fundamentally based on the principle of gating. Gates, or regions, are drawn on fluorescence scatter plots and histograms to selectively focus on populations of interest. These gates can then be applied to view and analyze specific populations.
The first step in gating is typically to distinguish the cells based on their light scatter properties. For instance, subcellular debris can be distinguished from single cells by relative size, estimated by forward scatter. Also, dead cells tend to have lower forward scatter and higher side scatter than living cells. Lysed whole blood cell analysis is the most common application of gating, and Figure 13 depicts typical graphs for SSC vs. FSC when using large cell numbers. The different light scatter signals of granulocytes, monocytes, and lymphocytes allow them to be distinguished from each other and from cellular debris.
Figure. 13. Analysis of lysed whole blood. A, SSC vs. FSC density plot; B, SSC vs. CD3 FITC fluorescence density plot. FITC, fluorescein isothiocyanate; FSC, forward scatter; SSC, side scatter.
Data can be analyzed as histograms or in two-parameter dot or density plots. On a density plot, each dot or point represents an individual cell that has passed through the instrument. The plots in Figures 13A and 13B are color intensity plots and are the most common way to represent a density plot. Here, the red/yellow/green/blue hot spots indicate increasing numbers of events resulting from discrete populations of cells. The colors give the graph a three-dimensional feel. With a little experience, discerning the various subtypes of blood cells becomes relatively straightforward.
While not shown in this guide, another less common way to represent a density plot is by a contour diagram. The graph takes on the appearance of a geographical survey map, which in principle closely resembles the density plot. It is a matter of preference, but sometimes discrete populations of cells are easier to visualize on contour diagrams.
In Figure 13B, the same cells are now plotted as SSC on the y-axis vs. CD3 fluorescence on the x-axis. CD3 is a marker that is expressed on T lymphocytes but is absent on other white blood cells. This highlights the usefulness of gating strategies that combine a scatter parameter with a fluorescence parameter.
Chapter 2: Compensation Controls
Single-Parameter or Univariate Histograms
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