Gating in FACS

Gating will allow you to view cells of interest by any combination of criteria that you choose. Gating does not change the intensity value assigned to an event as is the case for changes in voltage or compensation.

It simply lets you decide which data to view and which data to ignore or discard.

It is important to check that small changes in your gates don't have significant effects on your results or else your data will be prone to artifact.

When you create or format a data plot (i.e. on the Data menu select "Format Histogram" or "Format Dot Plot") you can select any of the gates that you have created.

This will filter the data and plot will only display those events which meet the gate criteria.

Gating will not discard data unless you have requested this under "Acquisiton and Storage". Gating can subsequently be changed when you analyze your data without any loss of information.

To set up a gate you first draw a "Region" using one of the tools on the tool palette (there are four geometric shapes to choose from outlined with dotted lines). Note that the "Marker bar" (designated with an M) and the quadrant maker tool next do not define regions. They are used for statistical analysis only and can't be used to filter data like the regions/gates.

Regions which are commonly employed include: PI for DNA content: FL2 area vs FL2 width. This window is useful for gaiting out apoptotic cells (lower left quadrant) and doublets (a separate cloud with increased FL2 width). FSC vs SSC: This is useful for gating out RBC, myeloid cells etc, from blood or marrow. Each region that is setup automatically defines a gate (e.g. G1 = R1). To delete regions select "Region List" under the Gates menu. To combine regions into more complex gating criteria use the "Gate List" under the Gates menu. (e.g. G5 = (R1 or R2) and R3).