On occasion you may need to constrain your G1 or G2 peaks in order to make it easier for FlowJo to fit the data. The constrain default is "Unconstrained".
When you open the Constraints disclosure (shown at right) you can constrain the peak positions relative to each other. Choose to constrain your peaks based on the Mean or CV and then select from the associated drop-down menu. For example, you can define the G2 peak position to be 2x the G1 peak, or the G1 peak to be 0.5x the G2 peak, etc..
If the fit is still undesirable, consider constraining the CV of the ill-defined peak. If the CV's for the two peaks are significantly different, then you should probably perform this constraint anyway. For most analyses, the G2 CV should be roughly equal to the G1 CV; you can set either one to be equal to the other, relieving that parameter from the fitting model.
You can also edit G1/G2 curves via click-and-drag. You'll notice that when a model has been selected, FlowJo produces vertical lines marked G1 and G2 on the graph. You can drag these lines to visually adjust these gates to the graph. Dragging vertically will include a wider range of values from the x axis, dragging horizontally will move the peak of the gate along the histogram line (during and after drag are shown at right).
To set Cell Cycle preferences, go here.
For information about the FlowJo Cell Cycle Models, go here.
For information about Cell Cycle Statistics, go here.
You may also wish to view the page on hints for performing Cell Cycle analyses.
Download a Cell Cycle Workspace with Demo Data to try out this platform.
