If these autogates totally miss the mark, and you’d like to define them from scratch, you can do so in FlowJo’s workspace, then click the control population columns to the right of the parameters, and select your manually-defined populations to assign them to that color. Now, adjust the range gate(s) there to your desired location. If the auto-gated control populations need adjustment, you can simply click on the preview graph windows at the bottom of the Compensation Wizard to bring up a univariate histogram defining these populations. Are the autogates correctly defining negative and positive control populations? If samples are mismatched, or no sample was selected for a given parameter, click the parameter cell to the right of the parameter column and select the appropriate sample.ģ. Are the samples assigned to the correct corresponding colors? If your parameter is not on this list, this is a sure sign that the parameter of interest is not contained in each single-stain control sample within your workspace’s Compensation group, which is required in order to perform compensation calculations on a given parameter.Ģ. If there are parameters which were not automatically added to the wizard, click on the parameter column header, and find the option “Choose Parameters.” Within the resulting dialog box, select your parameters of interest. If there are parameters included that you do not wish to compensate, click on the Sample cell to the right of the unwanted color, and choose “Remove this Parameter.” Are the parameters in the Compensation Wizard correct? Users should now be very careful to check the following three items:ġ. Then, peak-finding algorithms will try to auto-gate negative and positive control populations within those samples. Opening the Compensation Wizard.Īt this point, FlowJo will perform some pretty cool magic tricks: First, it will attempt to automatically assign control samples to their appropriate colors.
The Compensation Wizard in FlowJo is one of the most frequently used platforms, and by extension potentially the greatest source of confusion on a per-cytometrist basis.
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