MultiGraph Overlay

FlowJo can display multiple subpopulations of a single sample overlayed on the graph of the parent population for visual comparison. 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, convert it to an overlay graph and create gates directly with the Edit Gates tool in the Layout Editor .

To begin, drag a sample from the Workspace into the Layout Editor. Right-click the graph and select Make MultiGraph Overlay from the drop-down menu. If this population was previously gated, you can drag those gated subpopulations from the Workspace and drop them on the graph.

You can also create new gates in the Layout Editor by right-clicking and selecting Edit Gates from the drop-down menu. The display will change to the one below...

Here you can choose from three gating tools; rectangle, polygon or ellipse, using the icons above the graph. These function in the same way as their counterparts in the Graph Window.

The selection tool is active by default.

The X shaped icon cancels gate drawing.

 

After drawing a gate with the elipse tool, your graph will look like the one on the right. You can continue to add gates with any of the available gating tools. Each gated population is added to the legend with its subset name and color listed.

NOTE: New gates are added to the Workspace list as you create them. Each one appears beneath the population selected in the graph's layout legend. To make each new gate a child of the original sample, select its name in the legend before drawing the gate.

Click the colored square in the legend to change the color of the gated population in the graph.

Click and drag the Subset name in the Legend to move the population to the front or back of the graph display. (Z order)

If you wish to display more parameters of the same sample, you can add an additional graph to the arrangement in the Layout at any time. Right-click a graph, select the Multigraph Overlay menu item, then Add Graph.

FlowJo will try to choose parameters for the axes that are not already displayed. If you want to change the parameters displayed on the new graph, right-click the axis label and find Axes in the drop-down menu. There you can select from a list of all parameters available in the sample.

You may wish to display a whole set of overlays that show a plot of every parameter in relation to a particular one, or each fluorescent parameters vs. each of the others, or all available bivariate combinations. These options are all available with one click from the Add graph sets menu item.

At right is the Add graph sets menu item from a sample with five stain parameters. Selecting any of these items will add the named set of graphs to your layout all at once. Each graph will show the subpopulations overlaid with the color coding you have assigned in the legend.

Below is a graph set created by selecting All parameters vs. OrthSc. Each new graph displays the gated subpopulations overlaid in the color of the legend.