FACS (Fluorescence-activated cell sorting NOT cyTOF),Automating the removal of dead and duplicated cells

Question

[ccbb7aab4]

I have a large FACS project involving hundreds of files. So, it is not practical or suitable to manually gate these in Flowjo etc and to remove dead and duplicated cells. I have found many different methods and packages for cyTOF automation of these steps. I have yet to see any packages for FACS for these steps. I know there is a substantial difference in how you approach these two methodologies during the pre-processing steps. So I don't wish to use cyTOF packages.

Does anybody know of any packages that are useful for these steps? Or how to approach it from a novel standpoint.

Thanks in advance,
ccbb7aab4

[rfran010]

How are the samples related? Usually you just use the same gates for related samples.

Have you checked for FlowJo plugins?

[ccbb7aab4]

I have a healthy donor control for each run and then different samples within that run. The runs span almost 12 months. So, there will be technical variants that need to be considered. I have around ~1000 files that I need to deal with. I've not looked at FlowJo plug-ins yet. Do they have an automated approach? I know from past experiences with smaller cohorts that such a large timespan will inevitably introduce differences in how they sit in the space, so using the same gates won't be appropriate.

[rfran010]

I see. Yes, I know they have a plugin to sort of normalize those types variations, but I think that still requires manual determination of the control populations for each set. I haven't really looked into it otherwise.

[ccbb7aab4]

So with the manual determination of the controls for each run, can these be safely used as the gates for the samples for that run? Or will it be the case of having to manually gate on every one of the ~1000 files, including HD and samples?

[rfran010]

I never used it, but from what I understand, you assign negative controls, and then it will normalize each batch so that consistent gates could then be used...

[ccbb7aab4]

This looks promising, so we can apply CytoNorm before any cleaning has taken place?

[rfran010]

I haven't used it, so I never looked that deeply into it.

I would be interested to know how it works out for you, though.