FlowJo's backgating analysis provides a tool to view the effect of every gate in the gating tree of a multi-generation gated population.
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 (i.e if you took the final gate and applied it to the each population above it in the heirarchy). Therefore, you can quickly scan to see if any one of your gates is positioned incorrectly.
The backgating analysis is useful because it reveals that the population of interest, which may be very small, could have a very different distribution from the main population.
How to Display Backgating
Create a subpopulation that has three or more successive gating operations. Only a subpopulation three or more gates deep can be viewed in the context of each of its ancestor populations.
Drag the name of the subpopulation you wish to backgate from the Workspace into the Layout editor. Right click on the graph that is displayed there. You will see the right-click menu shown at below. Select with Backgating.
Once the heirarchy is displayed, you can choose to Orient Horizontal or Vertical by right-clicking on the backgate display. You can also choose to switch to a Show Ancestry view from the same right-click menu.
You can edit the Fill Color, Line Color and Line Style properties of the Backgate display by double-clicking on the display. This will bring up an Object Properties dialog, from which you can make the desired changes.
Backgating Example
Above is an example of backgating analysis on an intracellular stimulation. PBMC were stimulated and stained for CD3, CD4, CD8, and cytokines. Backgating analysis helps validate gate positions. Here, a typical cytokine gating analysis shows that cytokine producing cells have a different distribution than expected for most T cells, and that the gates should have been widened in some cases to accommodate this (see arrow).
A typical gating scheme was used to identify T cells (CD3+, left), lymphocytes, then CD8+CD4- T cells, and then cytokine producing cells (far right). The backgating analysis shows how each gate affects the identification of events. In each graph, the dots shown in green are fully gated cytokine positive cells. The gray dots in each individual graph show how the final gated population (ie cytokine positives) would appear had that single gate not been applied. Thus, in the second graphic, it is apparent that some CD3+ T cells producing cytokine fall outside of the typical lymphocyte gate (particularly, some appear as "blasts" at higher FS values). This analysis shows that the researcher might benefit from using a larger lymphocyte gate 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.
Note that the Layout Editor can also show a version of the backgating analysis, by double clicking on the graph to open the Layout Item Definition. Choose the "Show Ancestry" option and select the "Show Backgate analysis" option.
