Just run a single-color sample for every color in your panel and you'll be able to compensate your experimental samples in FlowJo. Thus, for that sample, if there is PE signal, then it must be spillover, and will be subtracted out by the software. The key thing here is that your compensation controls are single-color - so for example if you want to compensate FITC out of the PE channel, you need a "FITC only" control that the software "knows" only contains FITC. Since your single-color compensation controls have to be acquired and analyzed in order to generate the compensation matrix, they are not themselves compensated at the time of acquisition (i.e., you can't apply a matrix that you haven't already generated!).
I do this routinely with a 10-color panel. It will absolutely work to collect your compensation controls and samples without compensation applied and then generate and apply a compensation matrix in FlowJo post-hoc.
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