BiExponential Transformation - Instructions
The display transformation can be used on any compensated data--but FlowJo needs to be the one to compensate the data. Therefore, it can be done on any data where you collect the single stained compensation controls and create the compensation matrix, or, alternatively, on any data files which specify their own comp matrix (currently, only BD DiVa files do this correctly).
Software Compensation
To determine how to create a compensation matrix in FlowJo click here or download the Compensation Tech Note. Briefly, define (gate on) a positive and negative population on each single stained compensation control. Open the Open Compensation/Derived Parameters Editor (under the Windows menu) and drag the positive and negative populations from the workspace window onto the appropriate boxes in the dialog for each parameter. Click compute matrix. Apply this matrix to the experimental samples.
Procedure
(1) After creating the compensation matrix, double click on a stained sample (it may help to click on a gated subset of the sample that has the autofluorescence characteristics of the cells you wish to analyze.) This opens a graph of that tube. The axis labels are drop-down menus. Choose a compensated parameter from the menu.
(2) The Apply Transform icon appears next to the axis label. Left click and, voilá, the parameter on that axis will be displayed with transformation applied.
(3) Note also that the icon will change into a letter T that is not crossed out (below) and the transformation factor will be displayed.
Any compensated parameter displayed in a graph can be displayed with this Transformation applied by clicking the Apply Transform button.
You can adjust the Transformation settings by right clicking the Transform button.
Choose Change Transform Values from the pop up menu. This will open the following dialog...
