Research Techniques Made Simple — FLOW CYTOMETRY Q&A

RTMS-FlowCytometry.jpg

These questions and answers relate to the Research Techniques Made Simple article on FLOW CYTOMETRY by Richard R. Jahan-Tigh, Caitriona Ryan, Gerlinde Obermoser, and Kathryn Schwarzenberger in the October 2012 issue of JID.

We welcome your comments on the article and the quiz!

QUESTIONS:

1. Side Scatter (SSC) and Forward Scatter (FSC) provide information on ___________ and _________ , respectively.

A. tissue architecture, granularity
B. granularity, size
C. size, cell-cell interactions
D. cell-surface markers, intracellular signaling

2. “Gating” refers to:

A. the process of cells lining up single-file before entering the laser path.
B. the field the cells enter during the sorting process.
C. the restriction of a portion of the analyzed cells for further analysis.
D. the overlapping fluorophore signals generated in flow experiments with many fluorophores.

3. In a fluorescent by fluorescent scatter plot, cells present in the upper right quadrant of the plot are generally:

A. negative for one marker, positive for the other.
B. negative for both markers.
C. positive for aberrant marker expression.
D. positive for both markers.

ANSWERS:

1. B
Side scatter provides information that correlates to the cell granularity while forward scatter is a marker of cell size. With the aid of these two parameters and a few other stains, commercial blood analyzers are able to generate complete blood counts (CBC) with differentials. Remember, flow cytometry cannot provide information on tissue architecture or cell-cell interactions, as the cells must be suspended in solution for flow analysis. Flow cytometry can provide information on cell surface markers and intracellular signaling, but this is performed using fluorophore labeled antibodies, while measuring SSC and FSC does not require antibodies.

2. C
Gating refers to the process of selecting cell subsets of interest from parent populations during flow cytometry data analysis. For example, in a blood sample forward and side scatter can be used to define the lymphocyte region, and a second gate can be placed around CD3+ cells to discern them from CD3- natural killer cells. The field that the cells enter for a sorting is usually an electromagnetic field that separates based on charge. The overlapping signals generated from multiple fluorophores are termed “spillover”.

3. D
The interpretation of the basic fluorescent by fluorescent scatter plot is important, as virtually all flow experiments employ them. Cells in the upper right are positive for both markers, while cells in the bottom left are generally negative for both markers. The other two quadrants are negative for one marker and positive for the other as depicted in Figure 1ciii. Also, the axes for fluorescent by fluorescent plots are displayed logarithmically, meaning that even small visual differences reflect potentially large differences in fluorophore expression.

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