FlowJo v9 Manual


Derived Parameters

FlowJo allows you to add new parameters to your data. These parameters are not actually added to the original data files; they are simply descriptions of new parameters that are stored with the workspace. However, they behave in other regards like parameters that were collected: you can display them, calculated statistics on them, etc.

Because they are not stored with the data file, they will not appear if you open the same sample in a different workspace--you will have to add the derived parameters again in that workspace. Currently, there is no way to copy derived parameter definitions between workspaces.

There are four derived parameter types that you can create: time, ratio, log->linear, and linear->log. (Calibrated parameters are also derived parameters, but they are created using the Calibration Platform). To add a derived parameter, select the sample in the workspace, and then select the "Define new..." menu item attached to the "Derived Parameters..." menu item, under the workspace menu. You will be presented with a dialog window that allows you to specify the parameters to define. From this dialog window, you can also remove existing derived parameters. Click here to view the special derived parameter menu items.

Adding a time parameter is useful for performing kinetics analyses. This is usually done when you have a response that you wish to measure (for example, calcium flux experiments); however, you might want to see how stable a sample was during collection, and simply display forward or side scatter (or fluorescence!) vs. time during collection. Time can only be added if there is not already a time parameter with the sample. When you add a time parameter, FlowJo assumes that the event rate was constant throughout collection, and distributes events evenly over time. If the start time and end time for acquisition were recorded by the program collecting the data, then FlowJo knows the time over which to distribute the events. Otherwise, FlowJo will ask you to enter how many seconds it took to collect the sample.

You can add a ratio parameter: this is the ratio of any two collected parameters. This is commonly necessary for calcium flux experiments, in which the ratio of Indo-1 fluorescence in two different channels is related to the calcium concentration. You can choose to create a ratio parameter with either linear or logarithmic scaling.

You can create a calibrated parameter. Using the Calibration Platform you can convert the scaling of any parameter (for instance, to absolute number of molecules per cell). While the calibration platform creates a derived parameter automatically, you may wish to apply such a calibration to other samples--do this by selecting the appropriate derived parameter. You can directly select a calibrated parameter (once the calibration standard exists) by selecting it under the derived parameter submenu.

Finally, you can convert any collected parameter between log and linear. FlowJo will automatically optimize the scaling of the converted parameter to show the maximum number of events.

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