FlowJo computes the compensation matrix on control samples much the way you would manually set the compensation. To do this, you will select gates on positive and negative populations for each of these stain, and tell FlowJo to calculate the compensation matrix based on these stains.

Thus, you must collect samples that are singly-stained (as well as unstained) for each of the fluorescences that require compensation. Ideally, you would use a stain that only labels a portion of the sample population, so that you have an unstained set of cells in each tube. It is important to remember that the negative population and positive population must share the same autofluorescence characteristics for compensation to be valid (i.e., don't use monocytes as a negative control for a lymphocyte stain; if you are compensating a stain on fibroblasts, use an unstained fibroblast control).

If you need to generate another compensation matrix for other samples in the experiment, you can just repeat steps 2 through 4 as needed.

Once you have defined a compensation matrix, it is saved as a file on your Macintosh as well as in the workspace itself. You may subsequently apply that compensation matrix to other samples in the same workspace by selecting it from the menu; you can use that matrix in other workspaces by reading it in from the file that you saved. Note that a compensation matrix is generally valid only for samples collected during a single collection run!

Any sample that has been compensated is marked with a blue bar next to the sample name in the workspace window. Compensated samples have new parameters added to their list: for each fluorescence channel to be compensated, a new parameter is created. The parameter name is bracketed with "<>": e.g., when FITC and PE are compensated against each other, two new parameters named "<FITC>" and "<PE>" will be created. Remember to select these new parameters in the graph or statistics windows!