FlowJo gives you the ability to compensate your data. This may be necessary in cases where the compensation was inappropriately set during sample collection (although if the sample was over-compensated, then there is no recourse). Also, there may be cases where the instrument is not capable of compensating between certain channels (for instance, to correct for the spillover between FITC and Cy5PE).

For a description of why compensation is necessary, the underlying concepts behind compensation, and some of the pitfalls of improper compensation, see the "Compensation: A perspective" by Mario Roederer (internet connection required.) The remainder of the documentation below is NOT meant to teach you about compensation, simply how to use FlowJo to perform that function.

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

The matrix files are saved to a user specified directory whenever a matrix is defined. The files have an .MTX extension and can be edited with an XML or a text editor (for example notepad, wordpad..). Please note editing the matrix file is not recommended!

To perform software compensation, 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).

Steps involved in compensation by FlowJo are outlined in the Compensation Tutorial.

Any sample that has been compensated is marked with a 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 labeled with "comp-": e.g., when FITC and PE are compensated against each other, two new parameters named "Comp-FITC" and "Comp-PE" will be created. Remember to select these new parameters in the graph or statistics windows!

• An example of Compensation
• FlowJo's Compensation menus
• The Compensation window
• The compensation matrix file

Download a Compensation Demo Data to try out this platform.