![]() ![]() Multicolor background gates are best for identifying positive events. In FlowJo, 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. 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 assessing fluorescence spillover using compensation controls). 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 addition, in the multicolor world, sensitivity is often highly compromised by the post-compensation spread in autofluorescence distributions (see Compensation An Informal Perspective by Mario Roederer).Īn accurate way to identify positive events is the use of an "FMO" control (Fluorescence Minus One). ![]() However, discrimination is not so easy when the signal is low, when the positive population does not separate well from the negative. For many experiments, this is relatively straightforward, for example, when the signal is well-separated from negative cells. ![]() Nearly every flow cytometric experiment requires the discrimination of cells that exhibit fluorescence signal from those that do not. ![]()
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