Background
When a gate is drawn in the Cytobank platform, its size and location are the same across all files by default. Any subsequent changes to the size or location of the gate will apply to all files. Gates behave in this fashion when they are synced to all files and to all populations or global mode. Of course, it may be necessary to position or size a gate differently between samples, gating groups or depending on a certain parent population.
Click the links below to jump to the relevant section on this page.
- Global mode
- The Syncing menu
- Adjust gate location per file
- Adjust gate location per population
- Adjust gate location per file and population
- Adjust gate location to multiple files and/or populations
- Adjust gate location to gating groups
- Synced settings and the Boolean expressions tab
Global mode
By default, gates drawn in the Cytobank platform are in global mode. This means that the gates are synced to all files and all populations and that any change to their position or size will apply across all files and populations in the experiment. To set a gate to a specific location based on sample or population, see the sections below.
The Syncing menu
Gates syncing settings are always visible, represented by two icons on the right side of the Gates list. The first icon represents the gate Sync across files setting, and the second the Sync across populations setting. Synchronization can be set to all (arrow loop), within gating groups (arrow loop with a G on top) or to None (strikethrough arrow loop) across files or populations. Click on any of the icons to open the Sync menu. Use the dropdown list to modify gate position settings across files and populations.
(On the left, gate sync status is always visible and represented by icons. An arrow loop indicates sync to all, an arrow loop with a G on top indicates sync within gating groups, and a strikethrough arrow loop indicates sync set to None for files or populations. On the right, the Sync menu allows you to modify the gate position across files and/or populations.)
Click on Learn more on the upper right corner of the Sync menu to open the in-app help displaying how the different sync modes work without impacting the gate settings.
(In-app help shows gate position sync modes and the effect of modifying them. Change the settings here to see how the different sync modes work without impacting the gate settings.)
Adjust gate location per file
In some cases, it may be necessary to set a different gate location or size between samples. Consider the following example where a gate synced to all files falls in the wrong location on tight population between files:
(Gate placed correctly.)
(Gate placed incorrectly.)
Modifying the gate synchronization across files mode allows you to set the position of the gate differently between samples. To do so, select the gate from the list of Gates, click on the Sync icon and change the Sync across files setting to None. Finally, adjust the gate as necessary. As the gate position is synced to none of the files, the movement will only apply to the file being viewed. The result is shown below:
(Gate after adjusting the position per file.)
Remember to use the check gates functionality to view your gate location across all files!
Adjust gate location per population
During the gating process, it may be necessary to set the location or size of a gate on a certain parent population. A gate can be synced to all files and none of the populations, so the position is the same on all files, but it appears in a different place depending on the parent population.
Consider the following example: you want to analyze the percent of pSTAT3 response in CD4+ T cells and CD33+ monocytes. This figure can’t be made with just a global gate because the background pSTAT3 expression is very different between the two populations:
(The global gate used to capture percent of positive pSTAT3 can’t be set to accommodate both parent populations CD33+ monocytes and CD4+ T cells.)
To solve the problem, the gate synchronization across populations should be modified in the Gating Editor. To do so, select the gate from the list of Gates, click on the Sync icon and change the Sync across populations setting to None. Nudge one of the corners of the gate to link the position to the parent population. Finally, adjust the gate as necessary to the rest of parent populations. As the gate position is synced to none of the populations, the movement will only apply to the population being viewed. Moving the gate while visualizing a different population will generate a new child population incorporating the gate if such population didn’t exist already.
(Gate after adjusting the position per parent population to reflect correctly pSTAT3 expression in the CD33+ monocytes and the CD4+ T cells.)
Once the gate is synced to none of the populations, you can create new populations with a unique gate position by dragging and dropping the gate to a new parent population or using the Copy to population option from the gates hover menu to transfer the gate to a new parent population(s). When dragging and dropping or copying a gate that is synced to None populations, the correlate population will show in the Population tree and Boolean expression editor. This population is appended with the parent population in parentheses.
(The new child population refers to the pSTAT3 gate under the CD4+ T cells population.)
Adjust gate location per file and population
When a gate position is set differently for each sample, it may also need to be set differently to certain parent populations and vice versa. To allow this, select the gate from the list of Gates, click on the Sync icon, change the Sync across files and Sync across populations settings to None and nudge one of the corners of the gate to link the position to the parent population. After this point, any movement of the gate applies only to the file and population that are currently viewed.
Adjust gate location to multiple files and/or populations
A gate location may need to be set in batches, whether that is to batches of files or populations. The Copy gate to functionality opens a dialogue for accomplishing this. First, select the gate, click on the Sync icon and change the Sync across files and Sync across populations settings to None (if it's not the setting already).. Then, you must nudge the gate slightly and click on the Copy to population icon accessible from the gates hover menu to open the Copy gate position dialogue. Finally, select the files and populations you want to transfer the gate position to and click Apply. This will generate a new child population(s) incorporating the gate if such population(s) didn’t exist already.
(Click to open the dialogue to copy gate position to other files and populations.)
(The Copy gate position dialogue allows you to transfer the current location of the selected gate to other files and populations as needed.)
Copying a gate to multiple parents easily
A common workflow in cytometry analysis is to take one set of gates and apply them to other populations for quantitative analysis. You can accomplish this workflow in the Cytobank platform by copying populations in the Boolean expressions tab or dragging and dropping one gate into multiple populations one by one. However, there is an easier and faster option to batch apply these settings using the Copy to population tool. The workflow is visualized below:
(Illustration of workflow: copying gates to many parent populations.)
Select the gate, click on the Sync icon and change the Sync across populations settings to None. Then, you must nudge the gate slightly. The corresponding population is renamed indicating the parent population between brackets. See the effect using pSTAT5+ as an example:
(After setting Sync across populations to None and nudging the gate, the population is renamed indicating the parent population.)
Click on the Copy to population icon accessible from the gates hover menu to open the Copy gate position dialogue and select the other parents. The result for this example after copying the gate to the three other parent populations of interest is as follows:
(The pSTAT5+ gate has been transferred to other parents generating new child populations.)
Next, just repeat the process with the other gates to complete the workflow:
(The population tree at the end of the workflow.)
This workflow is the fastest way to create many descendant populations.
To keep the Population tree configuration and return the gates to global mode, simply select the gates from the Gates list, click on the Sync icon and change the Sync across populations setting to All populations. After globalizing the gates, the populations will remain, the gate will keep the same size and position for all populations as the one displayed when globalized. If you want to set a different position of the gate depending on the parent population, return the Sync across populations setting to None.
Adjust gate location to gating groups
When working with groups of samples or different panels, you may want to define gating groups that allow you to assign different gates and population hierarchies per group. Enabling gating groups in the experiment also allows you to set the location or size of a gate for the files and populations included within gating groups.
To do so, if the gate already exists in all the applicable gating groups, select the gate and click on the Sync icon to open the Sync menu. You can only set Sync across files to within groups if Sync across populations was previously set to None. Syncing the gate position to files within groups is also compatible with Sync across populations set to populations within groups.
(Gate position can be synced to files within groups and None populations (CD4+ gate) or populations within groups (CD8+ gate).)
After this point, any movement of the gate applies to all the files belonging to the same group and all populations (Sync across populations set to Populations within groups) or only the population that is currently viewed (Sync across populations set to None).
A gate present in different gating groups may have different syncing settings for each gating group. In the example below, the gate CD8+ is synced across files and populations within groups for the gating group named Pheno Basic and synced across files within groups and across None populations in the T cell gating group. This option grants you all the flexibility to complete the most advanced gating strategies.
(Gate position can be synced differently per gating group. The gate CD8+ is synced across populations within groups in the Pheno Basic gating group and synced across None populations in the T cell gating group.)
Alternatively, you can drag and drop the gate to a new parent population in a different gating group to create a new child incorporating the gate. Similarly, you can click on the Copy to population icon from the gates hover menu to transfer the gate to a new parent population in a different gating group. These actions will automatically update the Sync mode across files to Files within groups and Sync across populations to None. When dragging and dropping or copying a gate, the correlate population will show in the Population tree and Boolean expression editor.
(Dragging and dropping CD19+ B cells gate from B cell gating group to Pheno Basic gating group automatically set the Sync settings to files and populations within groups. The new population refers to the CD19+ B cells in the Pheno Basic gating group.)
Note that when transferring a gate from one gating group to another using the Copy to population option, if the gate syncing settings across files is set to All or Within groups, the dialogue will ask you to select which files and populations you would like to transfer the gate position to. If your file and population doesn’t match, for instance, you select files belonging to the Pheno Basic gating group and populations belonging to the T cells gating group, new populations will appear in all selected gating groups. Use the Undo button to revert to the previous state if necessary.
Synced settings and the Boolean expressions tab
The Boolean expression tab is used for various operations on populations, including adding and removing the populations and gates that define them. If a gate synced status across populations is set to Within groups or None, and it’s used to create new populations, independently of the method used, the new population definition expression cannot be altered in any way. This appears as the expression being lighter colors and not clickable. Please take this into account when creating your gating strategy.
(The expression of the populations cannot be edited in the Boolean expressions tab and appears in lighter colors if the transferred gate was Sync across populations to Within groups or None settings.)
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