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3D Clustering and Opening FCS files

Stephanie Foan (Author)

Thursday, February 2, 2012 2:03 PM


Anyone know if there is software that will allow me to cluster in 3D rather than sequentially gating on 2D dot plots? I'm staining for CD25, Foxp3 and CD127 and feel I lose out by looking at one molecule before the others then the other two separately. I know several let you generate pretty 3D graphs but can any software give you meaningful numbers?





Wednesday, March 7, 2012 2:03 PM

Hi Steph,

In fact I had the same problem as you. Now I analyse my samples with a programme that allows you to gate on 3D graphs and later to draw a region on 2D dotplots. This software has a  n-dimensional diagram (APS) where you can also select your events. It is very useful, if you dont know it, I would recomment you. I work with 8 colors so APS helps me a lot to separate the populations depending on sample parameters. The software is called Infinicyt.


Graham Pockley

Thursday, March 8, 2012 7:08 AM

Dear Steph

You might like to know that an evaluation copy of the Infinicyt software can be downloaded from here:

All the best

Graham (on behalf or the Chromocyte Team)

Stephanie Foan (Author)

Thursday, March 8, 2012 1:13 PM

Thank you both, I'll give it a go!

I hadn't actually realised until a closer look that FlowJo has a clustering function, but it's pretty poor and difficult to use, and only gives you a 2D gate you can use at the end of it all, as it sounds like Infinicyt does. I think Weasel is the same. I had been playing around in FlowJo and realised that one other thing you can do is export FCS files to .txt and open them in whatever you like. So if you're familiar with MATLAB, you can use any of it's clustering functions and generate 3D plots you can rotate etc etc.

What's really cool about the FlowJo exporting method is that you can use the autocompensate function in FlowJo, do a bit of preliminary gating, e.g. gating CD4 lymphocytes or something, then export only that portion of the file WITH compensation! So when I import the data into MATLAB a) it doesn't take for ages and b) I only need to write about 4 lines of code. Happy days!

Thanks again,



Friday, March 9, 2012 5:34 PM

Hi Steph,

I am an Infinicyt user. The APS graph that Ana told before is a Principal Component Analysis graph. You can display different views for the different components and get your markers listed by significance (that is, by its contribution to clustering). In addition, you can display a 2D APS or 3D APS in case you need to rotate the view to get a better perspective, amazing for my papers :)


Best wishes,