FlowJo v9 Manual


Compensation Matrix File

When the "Compute" button in the compensation matrix dialog window is clicked, FlowJo generates a compensation matrix. If you save the matrix to a file (using the "Edit/Save Matrix" option), the file looks like this:

Fluor PhyEry Cy5PE
1.0000 0.1803 0.0404
0.0091 1.0000 0.2595
0.0045 0.0473 1.0000

The first line has the title of the compensation matrix. The second 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).

Click here for instructions on modifying the matrix. In version 4.5 and after, the matrix can be modified right in FlowJo using the "Edit/Save Matrix" menu option. When you modify the matrix, the numbers are converted to give an estimation of the amount (percentage) of spillover into the other parameter.

Go back to the overview of compensation.

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