You can freely change the spillover coefficients. To reload the file, however, you will have to change its name. FlowJo identifies compensation matrices by the name on the first line of the file (NOT by the filename itself). Be sure to change this to something else; FlowJo will not allow you to load a different matrix with the same name as one already loaded.

Once you have reloaded a compensation matrix, you can simply choose the samples and reapply the new compensation matrix. Of course, all statistics (and graphs) will be invalidated.

How can you modify the compensation matrix? Let's suppose that you find that the FITC to PE compensation is too low: in other words, cells stained with only FITC have residual PE signal. First, draw gates around the two populations and calculated the median fluorescences (FITC and PE) for each of these populations. Calculate the ratio of the difference between the PE signals and the FITC signals. Add one to this ratio. This number is the correction factor: multiply the spillover coefficient between FITC and PE by this value and replace the original coefficient by the result.

Manual alteration of the spillover coefficient matrices is not trivial, especially when more than a few parameters are involved. If you find such alterations necessary, then perhaps you should consider the quality of the compensation controls.