FlowJo Version 4 implements a new platform for displaying subpopulations of a single sample. Overlayed subpopulations are distinguished by color - the color of a subset allows easy assessment of its characteristics on multiple plots. In addition to being able to display previously gated subsets of cells in overlay plots, you can start with an ungated sample and create gates directly in the MultiGraph Overlay platform itself.

Click once in the Workspace window on the sample you wish to analyze. Choose "MultiGraph Overlay" from the Platform menu. This brings up a window similar to the one below.

Graphical display. From the Configure Graphs Pulldown there are two choices for laying out graphs. "Scatter and Fluorescences" displays as many graphs as are necessary to display each parameter once. "All Histograms" displays a single histogram for each parameter. If you wish to customize the display you can:

-Add graphs (click the Add Graphs button)
-Delete graphs (click on the graph and choose delete from the popup menu)
-Change graph axes (click on the axis label)
-Move graph to end or beginning (click on the graph and choose from popup menu)
-Display a fraction of the events (change the percentage in the box at the top right)
-Change the color of the selected subpopulation or of the background of all plots

Save the customized display by choosing "Name and save this setup" from the "Configure graphs" pulldown menu.

Subpopulation names. The names of the gated subpopulations are displayed in the MultiGraph Overlay window. These subpopulations can be sorted by clicking the Sort button (FlowJo will sort the subsets in a hierarchical manner with the subset containing the most events at the top of the list). In addition, you can highlight a selected subpopulation by checking the box.

Creating gates on a MultiGraph Overlay. Select a population that you wish to create a gate on from the list (top center of the MultiGraph Overlay window). Click on a gating tool and create a gate in a graph window. The cells that fall in the gates subpopulation are displayed in color on all the graphs. Each subpopulation you create is displayed in the Workspace window under the sample name.

Overlaying previously gated subsets. Click once in the Workspace window to select the sample you wish to view as a MultiGraph Overlay. Choose MultiGraph Overlay from the Platform menu. Drag and drop a gated subpopulation node from the Workspace menu to the MultiGraph Overlay list. The cells that fall within this gate are now displayed in a different color.