How to create gates and populations

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Background: The difference between gates and populations
How to create gates and populations
How to rename/delete gates or populations
The impact of deleting gates or populations

Background: The difference between gates and populations

In the Cytobank platform, the terms population and gate are different, and the distinction is important to understand.

A gate is a shape drawn on a plot to encapsulate a set of events. It reflects the coordinates of the X and Y channels. It does not carry any other information such as hierarchy or complex cellular identity. For example, if you draw a CD4+ gate on a CD4 vs CD8 plot, that CD4+ gate corresponds to the coordinates of the CD4 and CD8 channel and does not define CD4+ T cells. To define the CD4+ T cells, we will need additional gates like CD3+ to define the hierarchical order by Boolean expressions.

To build information about hierarchy and to categorize cellular identity from gates, populations are used. A population is simply a collection of gates with certain Boolean expressions. In the example above, the population CD4+ T cells could be defined by the gates Scatter AND Live cells AND singlets AND CD3+ AND CD4+. This is the gate set of the population, and after applying all the gates, you will have the events of interest that define the actual biological population of interest. Since gates and populations are decoupled, it is important to understand how to create populations using gates and Boolean expressions.

The Cytobank platform allows you to work with groups of samples or several panels defining gating groups. You can define independent population hierarchies for each gating group by creating new gates or using common ones.

Gates can be created using the gating tools in the Gating Editor. The gates are listed without hierarchy, whereas the populations are listed in hierarchical relationships, with statistics in line.

You can create new gates and populations by:

Using sequential gating
Using drag and drop of an existing gate into one or multiple populations
Copying an existing gate into one or multiple populations
Copying a population hierarchy across gating groups
Defining new populations using the Boolean expression builder

The created gates will show on the left side of the plot listed in alphabetical order. The corresponding populations will show the proper hierarchy order nested under the proper parents and gating group, on the right side of the plot in the Population tree tab.

Sequential gating

During the gating process, you can do sequential gating to create populations. For example, you can complete the gate on CD3+ T cells and then create a CD4+ gate under CD3+ T cells population. This can be done either by choosing the corresponding population from the dropdown menu in the Population selection box or double clicking inside of a gate in the gating plot. Then, select the X and Y channels and the gating tool of choice to draw the new CD4+ gate. A new population with the same name as the gate will appear in the Population tree, nested under its parent population.

(Before drawing a new gate select the population from the Population selection box or double click inside a gate, then select the channels and the gating tool and draw the new gate. A new population with the same name as the gate will be created and displayed nested under its parent population in the Population tree.)

Drag and drop or copy a gate to a population

During the gating process, you can drag and drop or copy an existing gate to an existing population to create a new child population incorporating the gate info. For instance, you can transfer a pp38+ gate drawn under the CD33+ Mono population to the CD20+ B cells or CD3+ T cells. To do so, drag the existing pp38+ gate from the Gates list and drop it on the CD20+ B cells population to generate the pp38+ (CD20+ B cells) population.

(Drag and drop an existing gate into an existing population.)

Alternatively, copy the gate to a population by selecting the pp38+ gate name on the Gates list, hover over the CD3+ T cells population and click on Copy to population option to create the pp38+ population under that for CD3+ T cells.

(Copy an existing gate to an existing population.)

Use the Syncing settings to adjust the gate position to files and populations as needed.

Copy a gate to multiple populations

During the gating process, you can copy an existing gate to multiple existing parent populations to create multiple new child populations incorporating the gate info. For instance, you can transfer a pStat3+ gate drawn under the CD33+ Mono population to the CD20+ B cells, CD3+ T cells and CD4+ populations. To do so, select the Copy gate to option that appears when you hover over a the pSat3+ gate name on the Gates list and select the parent populations CD20+ B cells, CD3+ T cells and CD4+, to create the pStat3+ under CD20+ B cells, pStat3+ under CD3+ T cells and pStat3+ under CD4+ populations.

(Copy an existing gate to multiple existing populations.)

Use the Syncing settings to adjust the gate position to files and populations as needed.

Copying gates and populations across gating groups

When working with gating groups you can transfer one or multiple populations and gates from one gating group to a different one. To do so, select the population(s) you want to transfer and drag and drop them into the other gating group Population tree.

(Drag and drop population(s) to a different gating group.)

A prompt will ask you if you want to copy the missing gates between groups. If you select Yes, gates and populations will be copied to the selected gating group. If you select No, only the populations will be copied. If only the populations are copied and there are missing populations or gates in their definitions, a warning message will appear near the population name in the Population tree.

(On the left, prompt asking to copy missing gates. On the right, the effect of copying populations across gating groups without copying the missing gates.)

Read these articles to learn more about working with gating groups and syncing settings in the Cytobank platform.

Boolean populations

Complex populations can also be created using all Boolean operators (AND, OR, NOT and parenthesis) using the Boolean expression builder under the Boolean expression tab.

How to rename/delete gates or populations

To rename the gates or population, just mouse over the gate or population in the Gates list or Population tree and click on Edit to rename. Population names can also be edited from the Boolean expression tab. Please note that the gate name and population name are independent of each other. If one changes, then the other won’t automatically change. If a gate was created in one gating group and then copied to others, renaming the gate in one gating group will automatically update the name of the gate in the other gating groups. Populations belonging to different gating groups are independent and editing the name of a population in one gating group won’t change the name of other populations in other gating groups, however it will impact the sync group status.

(Use the Edit name tool to edit gates or population names.)

To delete gates or populations, just mouse over the gate or population in the Gates list or Population tree and click Delete. Similar to renaming gates or populations, deleting a gate won’t delete a population and vice versa. In the Population tree and Boolean expression tab, you can also select multiple populations to perform change of Prefix, Suffix, Duplicate or Delete with all the populations selected.

(Use the Delete tool to delete gates or populations.)

If you delete a gate present in more than one gating group, a prompt will ask you if you would like to delete the gate only in the group selected or in all gating groups. Please be aware that if the gate was used in the Boolean expression of other populations, deleting the gate may not be possible prior to removing it from all population definitions.

(On the left, prompt asking you to indicate whether you want to delete the gate only in the selected gating group or from all of them. The option is disabled if the gate is part of the Boolean expression of population(s) present in one (center) or multiple gating groups (right).)

The impact of deleting gates or populations

Gates and populations are different entities in the Cytobank platform. Deleting gates and populations can have different impacts depending on the Boolean expressions they are in.

When deleting gates, it is important to pay attention to where those gates have been used, as this might affect the corresponding populations.

If a population expression is defined by only one gate, deleting that gate will also result in deleting the corresponding population.
If a gate was used to define a population and that population is included in other population(s) expression, you must remove the gate from all population definition expressions before deleting the gate.
If a gate was used to define one or several populations using only the AND operator, deleting the gate won’t delete its correlated population(s), but the population expression and the hierarchy will be impacted and, therefore, no longer be the same population(s) you created before. Please double check the population hierarchy to make sure this is what you intend to do. If not, you can Undo deleting the gate.
If a gate is part of one or more population expressions using a Boolean operator other than AND, you must remove the gate from the population expressions before deleting it.

When deleting populations, similar scenarios apply:

If the population is part of other population expressions that involve only AND Boolean operators, the selected population will be deleted upon your confirmation, but the population expression of the remaining populations and the hierarchy will be impacted and, therefore, no longer be the same population(s) you created before. Please double check the population hierarchy to make sure this is what you intend to do. If not, you can Undo deleting the population.
If the population is part of other population expressions that involve Boolean operators other than AND, you must remove the population from the other expressions before deleting it.

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Table of contents

Background: The difference between gates and populations