Brief tutorial to compensating FlowJo's demo data

Create a new workspace. Add all four files from the compensation tutorial data.
Create a lymphocyte gate (FSC, SSC) and apply it to all samples.
In each of the control samples' Lymphocyte populations, define positive and negative regions using a histogram graph:

Once all the control samples have defined a positive and negative regions, click on the widnows menu, click on the sample to be compensated (or another sample with the same parameters) and open the "Define Compensation Matrix" window:

We now have to place the appropriate cell definitions in the appropriate square in the Compensation Definitions window.

To fill in the Fluor::aCD8/Negative, find its corresponding node in the workspace. Click on it once to select it. Then drag it with your mouse to the Fluor::aCD8 box in the Compensation Definitions window. A green icon should appear in the square, indicating a population has been placed there:

To clear any population from the Compensation Definitions, left-click on it and select "clear".

Once all the populations have been filled in properly, click "compute". This will ask you to save a compensation matrix file. We recommend to put this matrix in the same folder as your data - that way its easy to find. By default, FlowJo will put this file in your My Documents folder.

Click on the Lymphocytes in the test file (the file that needs to be compensated, 3-color.fcs)
Click Windows menu, assign compensation matrix. Navigate to where you saved the matrix file. Select it, click open.

Note the compensated sample has a green bar next to it.

The graph window will now have both compensated and uncompensated fluorescent parameters in the pulldowns:

Here is what the CD4 vs. CD8 graph window looks like before and after compensation:

[back to compensation]