Nearly every flow cytometric experiment requires the discrimination of cells exhibiting fluorescence signal from those that do not. For many experiments, this is relatively straightforward, for example, when the signal is well-separated from negative cells. However, discrimination is not so easy when the signal is low, when the positive population does not separate well from the negative. In addition, in the multicolor world, sensitivity is often highly compromised by the post-compensation spread in autofluorescence distributions (see Reference 1 below for more information).

An accurate way to identify positive events is the use of an “FMO” control (Fluorescence Minus One). This is a sample that has been stained with every reagent except for the one of interest; the difference between the FMO control and the test sample identifies positive events. In multicolor experiments, compensation-caused spreading in the distribution can be calibrated by using beads singly labeled with each fluorochrome (akin to the process of assesssing fluorescence spillover using compensation controls). In FlowJo Version 4, the Background Gating Platform uses the beads to compute an event-by-event background for each parameter, providing a way to discriminate positive events from those that look positive because of compensation caused spreading.

Multicolor background gates are best for identifying positive events. The “negative” populations will always contain a mixture of positive and negative events because of the inescapable overlap in distributions.

Procedure. In order to calculate the background in FlowJo, you need to compensate your data in FlowJo. Click on the "Calculate Background Gates" option in the Compensation dialog box. The calculation of background works best with single color compensation beads, but can be done with stained cells.

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