Limit / reduce the amount of events on the gating plot or normalize population event counts (slice of time)


Sometimes dot plots get too crowded and you want to reduce, limit, or otherwise downsample the number of events for visual purposes while gating.

Other times, you may have an experiment with many samples, but the samples all have different numbers of events and you want to standardize the number of events across the samples for analysis.

Gating on the time channel of your FCS files is a useful method for these situations and others. Click the links below to jump to sections in this article:

1) Limit / reduce the number of events on the plot while gating

2) Standardize event count across populations to control cell number

3) Clean up data acquired during a clog or machine abnormality


Limit the number of events on the plot while gating

In Cytobank, there isn't currently a special way of limiting the number of events that are shown on the plot by a percentage or a slider bar. Instead, a gate will have to be drawn on the time channel.

To "time slice" your data, navigate to the gating interface and change the X axis to time. The Y channel is irrelevant, but you will want to make sure to make the gate is tall enough to capture any variation within the Y dimension.


 (example of gating time and then activating the gate to reduce events. The gate is activated and deactivated serially (off screen) to show effect. The result is fewer events being shown on the scatter plot)

Note, you might have to adjust your scale settings to shrink or widen the overall time distribution.

Note, tailor the gate location per file to set the time gate to a unique location for different samples in your experiment.


Use the time gate to adjust or normalize event count of existing populations

If you incorporated a time gate into your original gating strategy, then your populations will already be using it. However, if your time gate is an addition after you have already gated, you will need to redefine your gating strategy to include the time gate. The beginning state of the population tree might look like this with the time gate outside the hierarchy:

(a population tree with a time gate that is not part of any gating strategy)


In order to make the time gate part of the other populations, the Population Manager must be used. See the animation below for how the time gate in the example above is added to the definition of each existing population using the Population Manager:

(a time gate is added to an existing gating strategy using the Population Manager)


Clean up data acquired during a clog or period of abnormal machine behavior

Scanning the time channel of FCS files is a good practice to make sure the data were acquired consistently from the machine.

Using a polygon gate on the time channel is a useful way to gate out periods of an acquisition that have resulted in questionable data. The polygon allows for multiple slices of time with a single gate. See the example below for taking only prime segments of data:


 (gate data on time with a polygon to take multiple slices of time in one gate)





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