The backgating analysis shows how each gate affects the identification of events. 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).Ī typical gating scheme was used to identify T cells (CD3+, left), lymphocytes, then CD8+CD4- T cells, and then cytokine producing cells (far right). Backgating analysis helps validate gate positions. PBMC were stimulated and stained for CD3, CD4, CD8, and cytokines. Backgating ExampleĪbove is an example of backgating analysis on an intracellular stimulation.

You can edit the Fill Color, Line Color and Line Style properties of the Backgating display by selecting the backgating image, click on the Object Tab and setting these attributes. You can also choose to switch to the Show Ancestry view from the same right-click menu. Once the hierarchy is displayed, you can choose to Orient Horizontal by right-clicking on the backgate display. You will see the right-click menu shown below. Right click on the graph that is displayed there. Only a sub population with two or more gates deep can be viewed in the context of each of its ancestor populations.ĭrag the name of the sub population you wish to backgate from the Workspace into the Layout editor.

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.Ĭreate a sub population that has two or more successive gating operations. Therefore, you can quickly scan to see if any one of your gates is positioned incorrectly. 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 hierarchy). FlowJo’s backgating analysis provides a tool to view the effect of every gate in the gating tree of a multi-generation gated population.įlowJo’s backgating option generates graphs within which each level in the gating hierarchy is displayed, with the final gated population overlaid at each level.

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