FlowJo's backgating analysis provides a tool to view the effect of every gate in the gating tree of a multi-generation gated population.

A subpopulation created by several successive gating operations can be viewed in the context of each of its ancestor populations. Drag the name of the smallest subpopulation from the Workspace into the Layout editor. Right click on the graph that is displayed there. You will see a drop-down menu shown at the right. Select Show Backgating.

FlowJo generates graphs within which each level in the gating hierarchy is displayed, with the final gated population overlaid at each level. This overlay is a backgating overlay: in other words, it shows the final gated population within the population of its ancestors. Therefore, you can quickly scan to see if any one of your gates is positioned incorrectly.

Above is an example of a simple backgating analysis. The CD8 positive population at the top is shown in red within its parent population (lymphocytes) and grandparent population (the full sample.)

The backgating analysis is so critical because it reveals that the population of interest, which may be very small, could have a very different distribution from the main population.