Gating allows you to progressively filter your data to isolate specific cell populations. For example, you can select singlet cells based on scatter parameters, then select T cells, B cells and other immune cell populations based on expression of phenotyping markers.
- Set your plot channels to the markers that you want to use for gating.
- Click one of the gate buttons above the plot: rectangle, polygon, ellipse, range, quadrant or split. (Range and split gates require setting the plot type to “histogram.”)
- Draw the gate on the plot.
- Name the resulting population when prompted. The population will appear in the hierarchy.
- To view the events inside of the gate, double click the gate on the plot, or click the name of the gate in the population hierarchy.
Coloring plots by a third channel¶
Coloring a plot by a third channel can assist with gating.
- Change the Plot Type to Dot. A Color Channel option will be added to Plot Settings.
- Click on Color Channel and choose the channel to color the plot.
Coloring plots by a third channel can also be done when making illustrations.
By default, all FCS files in an experiment will use the same gate geometry. In some scenarios, you may want to adjust a gate to a specific file or set of files. For example, if your experiment includes samples collected on different days or from multiple donors, your gates on the scatter channels may need to be adjusted.
Generally, avoid tailoring a gate when statistics from that gate are used for comparisons between files. For example, if you are comparing the median fluorescent intensity of pSTAT5 in a population between two files, you should use the same gate geometry for both files.
Creating tailored gates¶
- Select a gate to tailor.
- Click the tailored per file switch to enable tailoring.
- Adjust the gate as necessary.
A tailored version of a gate is only created for an FCS file when a gate is first moved after enabling tailoring. Thus, any files for which you have not adjusted the gate since you enabled tailoring will continue to use the default gate.
Applying tailored gates to other files¶
After you have tailored a gate to a file, oftentimes you will want to use that same tailored gate geometry for other files. For example, you may want to use your viability gate for all files from the same donor. You can apply tailoring using either file annotations or filenames.
- Select the FCS file and gate from which you want to copy the gate geometry.
- Click the apply tailoring to… button.
- Either manually select files from the list; or select annotation criteria to match, select the matching mode (any or all) and click select. You can view the selected files and refine the selection in the file list.
- Click apply. A message will be displayed indicating to how many files the gate was applied.
Changing a gate from tailored to global¶
When you turn off tailored per file mode for a gate, the gate geometry that is currently displayed will become the global gate that is used for all files.
Resetting a tailored gate for a file¶
You can reset a file to use the global gate instead of its tailored gate.
- Select the FCS file for which you wish to reset the gate.
- Select the gate that you wish to reset.
- Click the reset tailored gate for file button.
Copying and Pasting Gates¶
You can copy gates to different parts of the gating hierarchy using the population hierarchy menu.
- In the population hierarchy, right-click or click on the three vertical dots next to the population that you want to copy, and select copy.
- Right-click or click on the three vertical dots next to the the population where you want to paste, and select one of the paste options. See below for a description of these options.
|Paste with children||Pastes the population with its children.|
|Paste without children||Pastes only the copied population, without its children.|
|Paste children||Pastes only the children of the copied population, without the copied population itself.|
|Paste … (linked)||These options copy the population(s) as above, but the new populations are kept synced with the copied populations. Modifying either the original or the copied gate will modify the other as well.|
|Paste gate geometry||Pastes the gate geometry (shape) from the copied gate. This modifies the selected gate so that it has the same shape and position as the copied gate. It does not create a new population. The source (copied) and target (paste-to) gate must be the same type (e.g. both polygons). If the target gate is tailored, the change is applied to the current file only.|
Copying and pasting gates preserves tailoring. That is, if your copied gate is tailored, those file-specific tailored gates will be pasted.
Paste gate geometry and paste … linked can be useful when you have signaling, cytokine or subsetting gates that you want to have the same shape and position below multiple parent gates. For example, you might want a Granzyme B+ gate to have the same position below both CD4+ T cells and CD8+ T cells.
You can easily import gates from another experiment.
Converting rectangle gates to polygon gates¶
If you drew a rectangle gate and you later want to change its shape, you can convert it to a polygon gate.
- In the population hierarchy, click on the three vertical dots next to the gate that you want to convert.
- Click Convert to polygon.
Adding and removing polygon points¶
To add a point to a polygon gate:
- With the gate selected, click on the three vertical dots next to the gate’s name in the population hierarchy.
- Click Add polygon point.
- Use your mouse to position the new point.
To remove a point from a polygon gate:
- Select the point that you want to delete. There should only be one point with an orange handle.
- Hit delete on your keyboard.
Skewing a quadrant gate¶
To skew a quadrant gate:
- Right-click on the gate name in the gating hierarchy.
- Select Enable skewing. If the gate is tailored, choose between Enable skewing (all files) or Enable skewing (this file only).
- Click and drag any of the nodes to a new position.
To disable skewing:
- Right-click on the gate name.
- Select Make orthogonal. For tailored gates, choose between Make orthogonal (all files) or Make orthogonal (this file only).
CellEngine supports creating populations using “not,” “or” and “and” logical operators.
- To create an “and” or “or” population, or to create a “not” population using more than one gate (“not any”/“nor”), ctrl-click to select the desired gates in the hierarchy.
- Right-click on a population or click on the three vertical dots next to a populations’s name in the hierarchy.
- Click Create “not” population, Create “or” population or Create “and” population.
Note that the CellEngine API supports complex populations constructed with any combination of and, or, not and xor operators. Only a subset of these populations can be displayed in the Web interface.
CellEngine can automatically create combinations of gates, ideal for analyzing cytokine expression. For example, if you have populations named “IL-2+” and “IFNa+,” creating combination populations from those will create four new populations: “IL-2+ IFNa+”, “IL-2+ IFNa-”, “IL-2- IFNa+” and “IL-2- IFNa-.”
- Select between one and 12 gates by ctrl-clicking or shift-clicking them in the hierarchy.
- Right-click and select create combination populations.
Locking Gate Positions¶
CellEngine provides two kinds of gate locking that prevent changes to gate positions. Locking can be useful for preventing accidental changes in long-duration experiments that you analyze over the course of multiple days, and in workflows with quality assurance steps.
Locking a specific gate for all FCS files¶
In the left panel on the gating page, checking the Lock Gate checkbox will lock the selected gate’s position for all files in the experiment.
Locking all gates for a specific FCS file¶
On the annotation page, checking the checkboxes in the Gates Locked column will lock all gates for the selected FCS files. When a file’s gates are locked, these actions are disabled:
- Modifying a tailored gate when viewing an FCS file with locked gates
- Modifying a global gate when viewing any FCS file
- The reset to global gate button when viewing an FCS file with locked gates
- Applying tailoring to an FCS file with locked gates
- Disabling tailored per-file mode when viewing any FCS file