FlowJo’s hierarchical gating method automatically designates AND Boolean gates by showing one subpopulation indented and underneath another in the Workspace window. The NOT Boolean gate is designated in the “Active Gate” area of the graph window by unchecking “gate events inside”.

You can now create a gate that is a Boolean combination of any existing gates using all the Boolean function, including “OR”.

To create or edit Boolean gates, choose the parent subset and select the menu item "Boolean gates..." under the "Platforms" menu.

Choose from the subset “Insert Subset” pulldown menu.

Choose the function from “Insert Function” pulldown menu.

Name the gate and click “Create”. The Boolean gate will be displayed in the workspace window as part of the gating hierarchy.

Boolean gates are "live", meaning that they will reflect any changes to the gates on which they depend. Boolean expressions can be arbitrarily complex; you can use parentheses to associate the expression properly. For example: “Live and (CD4 or CD8) and not GammaDelta” will create a gate that includes cells in the predefined “Live” gate, “GammaDelta” gate, and either the “CD4” or “CD8” gates.

Note that the Boolean expression you specify is applied to the cells in the subset to which you are adding the Boolean gate. For example, if you add a Boolean expression to the Lymphocyte gate, then only lymphocytes can be part of the gate (no matter what the Boolean expression is you can't include a Monocytes gate)—if you specify “not Lymphocytes” in the expression then you would get no cells! In this case, you probably want to add the Boolean gate at the sample level.

Because all parent gates are automatically part of the final gate (from FlowJo’s standard hierarchical approach), they will not appear in the popup menu of possible subsets to include in the expression.

FlowJo can also automatically create a series of Boolean gates that represent all combinations (plus and minus) of a set of gates. For example, if you have a sample which was stained for CD8 and CD4, you can automatically create 8 gates that represent all combinations of expression of the cytokines (e.g., CD4+CD8+, CD4+CD8-, CD4-CD8-) using FlowJo's Boolean Combination option.