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The file generated by FlowJo in response to the "Compute" button in the compensation matrix dialog window is a text file. For the example from the tutorial, this file looks like this (each line is terminated by a single carriage return only): HRH
960814 The first line has the title of the compensation matrix. The second line has the prefix and suffix that is applied to each parameter channel name to denote the compensated channel name. The prefix and suffix are separated by a tab. The third line has the names of the fluorescence channels that are being compensated. Channels that are not in this list are not compensated; channels in this list that are not in the sample are ignored. The names must match exactly; use the names that appear in the $PnN keywords of the FCS file. The names are separated by tabs. The subsequent lines are the spillover coefficients, corresponding to each of the channels named. The rows are the spectra of each channel; the columns are the contributions of each fluor into the channels. Thus, 0.1803 is the spillover coefficient between FITC and PE, or the amount of FITC signal in the PE channel for a FITC-stained cell; 0.0091 is the amount of the PE signal in the FITC channel for a PE-stained cell. The coefficients are tab-delimited. In very rough terms, the 0.1803 corresponds to approximately 18% spillover (PE-%FITC). When modifying the matrix file, do not change the formatting; make sure that the number of coefficients, names, and lines are correct as well as being tab-delimited. Go back to the overview of compensation. |
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