Compensation Example

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This is a simple example showing how to do three-color compensation in FlowJo. There are several steps involved.

1. Set a gate for the positive and negative populations for each singly-stained sample.
2. Bring up the Compensation Definition dialog window, or Compensation Wizard.
3. Drag each of the positive and negative populations from the workspace window into the appropriate box in the compensation dialog window.
4. Compute the matrix, save it onto disk.
5. Apply the compensation matrix to the appropriate samples.
6. Display the compensated parameter (in brackets <>) on the graph axes.

You can follow this example on your own computer by downloading the Compensation Demo Data.

In some cases, you will need multiple compensation matrices for the same experiment. This is true, for instance, when you are using different lots of tandem dyes (like Cy5PE). Different Cy5PE reagents (even from the same manufacturer) have different spectra and thus require different compensations. You will need to collect a singly-stained compensation control sample for each different Cy5PE reagent, and then construct a different matrix for each of these.

When making multiple matrices like this, you only need to repeat the drags of the successive Cy5PE populations followed by steps 4 and 5, saving a different matrix for each set of reagents.

The next page begins the tutorial.

Go back to the overview of compensation.