Hi Greg,
We'd be happy to do a conference call to walk you through whichever option you prefer. We are in Sydney, Australia (US east coast + 16 hrs) so we'd just have to line up a time. Shoot us an email at
thomas.ashhurst@sydney.edu.au if you want to set it up. However, before we do that, you should check out the resources available to help get you going.
PhenoGraph is an awesome tool, I'm a huge fan. Both the Cyt package in Matlab and the CytofKit package in R, described in the other comments, can run in a user-interface format (point and click, rather than actual coding) which is a little easier to use. There is still a bit of learning involved to get it to run properly, but it's not a huge threshold to get past. Then there is the scripted version that you can run in R using the package mentioned by Vinko. This is the 'better' way to do it, because you have a huge amount of flexibility in managing your data. Whilst it is a little harder to learn, it is a huge investment in data analysis in the future. There is also some other options out there, many of which are included in the CytofKit package. I've been particularly partial to FlowSOM these days for running really large datasets (
https://github.com/SofieVG/FlowSOM) but we run it in R. CytofKit contains FlowSOM code, but I don't think that it is implemented in the user interface component at the moment (though I might be wrong, anyone reading feel free to correct me).
If you download the Cyt package from Dana Pe'er's website (
https://www.c2b2.columbia.edu/danapeerl ... nload.html) there is a screenshot guide that comes with it. We also have a screenshot protocol at
https://sydneycytometry.org.au/computational-analysis/. Our current version is focused on running tSNE in Cyt, but many of the steps are in common. Because of the way we store it, you'll have to 'request permission' after clicking on the link. One thing you'll have to keep an eye out for in Cyt is a that in your FCS files, some parameters will come with a 'parameter' name but not a 'stain' name (such as event length, etc). These come up as blank stain names in Cyt which stop the program working properly. You can use the re-naming function in Zach Bjornson's R package (
https://github.com/nolanlab/cytofCore), or just avoid including parameters that would have the problem (event length, time, or FSC/SSC etc from flow data). We have some screenshots and an implementation of Zach's script if you want to try it -- it's not online yet, but I'll throw it up on
https://github.com/sydneycytometry later today. The CytofKit guide (
https://www.bioconductor.org/packages/3 ... ample.html) has a bunch of screenshots to walk you through using it, so that should be fairly straight forward.
Try these out first, and see how far you get. Feel free to get in touch in the meantime if you need help.