Compensation Scripting in Macintosh FlowJo 8 tutorial

Compensation scripting will take an existing compensation template and freshly calculate/apply compensation matrices based on the rules you define in the script. To follow this tutorial I've prepared some workspaces and files you can download:

script.zip

To get started, take your single stained control tubes and put them in their own groups.

Tip: make sure the groups are dynamic. What keywords can you use to differentiate your FITC comp control from all the other tubes in your experiment? Use those keywords in your group definition.

Then, design gating for each control tube and make it group owned by dragging the gate(s) to the proper group.

Tip: all gates which are not Group Owned will not be saved in templates. If you plan to make a template, make sure all your gates are group owned or they will be lost!

By now, your workspace should resemble the one in step 1 in the file attachment above, screenshot below:

 

Now, click Platform menu, Compensate Sample, "AutoComp Scripts...". You'll be presented with this interface:

 

I've named this script "demo script". The "define a rule.." checkbox is important: This setting controls how your samples will be compensated. In this example, $DATE (date of acquisition keyword) has to match controls and mixed tubes, otherwise compensation will not happen.

Tip: compensation controls should be acquired with every experiment for best results. If you are "recycling" older compensation controls, you might as well do compensation by "eye" (and join the Pastafarians)

using the "Add Compensation.." button to add all fluors' controls:

 

above, I've already selected $FIL for the keyword matching. Pick a keyword unique to your control sample. Here's how I set mine up:

 

Once your controls are set with all 4 values (keyword, value, pos gate, neg gate) you can optionally choose the behavior from the bottom menu. I picked "only Uncompensated samples" :

 

Then click Done.

Then click File, Save as Template.

This way when you load the next experiment into this template, the following things will happen:

1) compensation tubes will be recognized by the template, put into their own group, to inherit their proper gates.
2) once the gates are in place, compensation script will evaluate the gates and determine spillovers from the fresh samples.
3) compensation (purple bar next to file name) will be applied to any files that match the script definition.

Comments

[RS]

I saw a useful tip on ur blog (making multiple graph sets , sep 26 ) and I cant get it to work. I dont see those options when I control+ click in the l.ayut editor on mhy mac. What am I doing wrong???????
Thanks , ;)