Background
When a gate is drawn in Cytobank, its size and location is the same across all files by default. Any subsequent changes to the size or location of the gate are applied to all files. Gates behave in this fashion when they are in global mode. Of course, it may be necessary to position or size a gate differently between samples, or depending on a certain parent gate / population. When a gate is not in global mode, it is in tailored mode. A gate can be tailored per file, tailored per population, or both.
Click the links below to jump to the relevant section on this page.
- Global mode
- Tailored per file / sample
- Tailored per population
- Tailored per file and per population
- Applying a gate tailoring to multiple files and/or populations
- Tailoring per population and the Population Manager
By default, gates drawn in Cytobank are in global mode. This means that any change to their position or size will apply across all files in the experiment. To set a gate to a specific location based on sample or population, see the sections below.
It may be necessary to set a different location or size for a gate between samples. Consider the following example, where a global gate falls in the wrong location on a tight population between files:
(file 1 - gate placed correctly)
(file 2 - gate placed incorrectly)
To set the location of the gate differently between files, select the gate, click the tailored per file button, and then move the gate. The movement will only apply to the file that is currently being viewed. The result is below:
(file 2 - after tailoring the gate location)
Remember to use the check gates functionality to view your gate location across all files!
Tailoring a gate per population (TPP)
It may be necessary to set the location or size of a gate depending on certain parent population. When a gate is tailored per population, it appears in the same place on all files but appears in a different place depending on the active population. Consider the example below:
I 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:
(with just a global gate, the gate to capture percent positive can't be set to accommodate both populations. The gate needs to be tailored per population)
To solve the problem, the gate is toggled into tailored per population mode in the gating interface, then each population is chosen and the gate is dragged to the correct location for that population. The result:
(the issue illustrated in the previous figure is solved. This figure is easily made with a gate that is tailored per population. Note the location of the gate is different between the monocytes and CD4+ T cell columns)
When working with gates in TPP mode, correlate populations will also appear in the population manager and population tree in the gating interface. These special populations are designated with the parent they are tailored to in parentheses:
(A population to reference the pSTAT3 gate that is tailored to CD4+ T cells can be seen above. The populations also appear in all the expected places throughout the Cytobank Experiment)
Tailoring a gate per file and per population
When a gate is tailored per file, it may also need to be tailored per population, and vice versa. To enable this, simply click both tailoring buttons in the gating interface while the gate is selected. After this point, any movement of the gate applies only to the file and population that are currently selected. With an understanding of both principles on their own, it is naturally understood how gates behave with both tailoring modes enabled.
Applying tailored gates in batches to groups
A gate location may need to be set in batches, whether that is to batches of files or populations. A dialog for accomplishing this can be opened from the gating interface:
(click to open the dialog for applying tailored gates)
(the simple dialog allows applying the location of the current gate to other files and populations as relevant based on the tailoring mode)
Using batch apply to transfer child gates to multiple different parents easily
A common workflow in cytometry analysis is to take one set of gates and apply them to other populations for quantitative analysis. The typical way of accomplishing this workflow is by copying children gates with the population manager. However, the tailoring per population (TPP) functionality and the ability to batch apply these settings (see section above) offers a fast and easy alternative way to accomplish this workflow.
The workflow is visualized below:
(illustration of workflow: copying children gates to many parents)
Copying these gates and populations through the population manager can be time consuming and tedious if there are many gates to be copied. Instead, select a child gate within the gating interface and tailor it per population. After tailoring it per population, nudge it slightly in order to solidify its tailored state. The following effect will happen (using pSTAT5+ as an example):
(after tailoring a gate per population and nudging it - it indicates which population it's tailored to)
Once the gate is tailored per population, use the apply dialog as described above to apply its tailoring to other parent. The end result for this example after applying to the three other parent populations of interest by clicking apply tailored gate and choosing Monocytes and the two T cells populations:
(the pSTAT5+ gate has been applied to other parent populations)
Next just repeat the process with the other gates and the process will be complete:
(the population tree at the end of the workflow)
This workflow creates many TPP populations and gates. To keep the population tree configuration and return the gates to global mode, simply select the gates in the list of gates and switch them into global mode with the tailoring switch (illustrated in sections above). After globalizing the gates, the populations will still remain and the core workflow will be accomplished but it will appear as though tailoring per population was never used.
The Population Manager and tailoring per population
The Population Manager is used for various operations on populations, including adding and removing the gates that define them. Certain operations in the population manager will be blocked when working with gates that are tailored per population.
- The gate that is tailored per population cannot be added or removed from populations.
- When a population is created as a result of tailoring a gate to a population (pictured above), its gate definition cannot be altered in any way. This appears as the entire row in the Population Manager being gray and not clickable.
If this is holding back your workflow, please get in contact with Cytobank Support for product feedback and tips on workarounds.