Graph Sets

Hey somebody reads this blog! One of our users has read this post (discussion of the same topic for the PC version) and asked,

how does one make graph sets and multigraph overlays on the Mac version?

Well, both are possible but as you would expect - slightly different from the PC version.

Graph Sets:

Unlike the windows version, graph sets are done through graph windows. To get started, load some samples to your workspace, and open the Graph tab of your preferences:

and click on the "choose..." pulldown menu on the bottom left, to select "create new set.."

You can populate the X and Y axes by clicking on them one at a time. To produce a histogram, leave the Y axis blank.

hint: The options for X/Y axes are built from the data file(s) present in FlowJo's currently selected group.

Once you've defined a "graph set", click "save" and close the preferences dialog. Then, click-and-hold one of the samples in the workspace window:

the graph sets are listed near the top. Ours was called "Set 1". Clicking it from this menu will produce this display:

so now you've got a graph window for every X/Y definition in your "set".

Unfortunately this does not aid in creating hierarchical gates, only gates on the same level (siblings or booleans)

For hierarchical gating, we use the

Multigraph Overlay Platform
read the manual entry

Click on a file in your workspace and then click Platforms, MultiGraph Overlay. FlowJo will present you with a new window which has one graph of the first two parameters:

To add graphs, just click on "configure graphs" menu and select option(s) for what graphs to add:

Now we have a few different graphs showing different populations:

You can add/remove plots at will. If you wanted to add a histogram of CD3, click Add Graph button, then click on the new graph's  X/Y axis labels to change them to "cd3" and "histogram" respectively:

Let's begin by making a CD3+ gate:

hint: the gating tools are currently grayed out. To enable them, click on a "layer" of what cells to gate on in the upper center portion of the Overlay window.

hint: to gate on histograms, choose the "rectangular gate" icon. We should probably get that fixed ;)

Notice that creating a CD3+ gate has made a new layer in the upper-center plot, and highlighted the CD3+ cells in green for all plots.

But, in our FSC/SSC plot we see that some of the CD3+ cells are falling outside the Lymph cluster. Let's make another gate - a subset of CD3+ - that will filter out the large cells. To gate o n only CD3+ cells, click on the CD3+ layer in the upper-center portion and then create a gate:

So, we've made some simple hierarchical gates using this example. What does this look like in the workspace?

Well, in addition to the usual gate tree, there is one additional "node" - the multigraph overlay. This stores the types of graphs you were viewing and coloring info etc..

Hint: you can attach the Multigraph Overlay nodes to groups and save your workspace as a template so that you can always dive back into the MGO window if you need to.

 

Comments

[RS]

Thanks for the multiple graph sets on mac tip. And I do check out this blog regularly (ok, thats a lie.. only when i have problems). I have a problem with a 7 colous stain and biexponential gating right now , is there a place where i can upload a powerpoint which shows what the problem is?

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Hi, I've emailed you twice, but I don't know if you got my emails. You can contact me at simm@treestar.com