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p-STAT5: Cytof vs. Calibur

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ianfrank

Contributor

Posts: 29

Joined: Thu Nov 21, 2013 11:59 pm

Location: Tampa, FL

Post Tue Dec 24, 2013 12:30 am

p-STAT5: Cytof vs. Calibur

Dear All,

I've recently started phosphlow experiments on the Cytof. I'm looking for p-STAT5 in human PBMC. The cells are stimulated with IL2 100IU/mL for 10 minutes. This always creates a huge p-STAT5 response of 20 to 60% of the CD4 cells. I've not been getting near the response by mass as I do by traditional flow. I've attached a pdf. The same tube(s) are split in 2 after stim, fix, and perm. Then stained for either mass or fc. Is this normal? Should I have lower expectations for mass positivity in terms of frequency?

Ian

stat5 mass vs. calibur.pdf
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maecker

Contributor

Posts: 23

Joined: Thu Oct 31, 2013 7:58 pm

Post Sat Jan 25, 2014 9:27 pm

Re: p-STAT5: Cytof vs. Calibur

Hi Ian,

Was hoping someone else would have a good answer for this. I would only have two comments:

1. I wonder if your pSTAT5 Ab for CyTOF is performing optimally. Is it a DVS conjugate or your own, and is it the same clone in fluorescence vs. CyTOF?
2. We of course don't usually count %+ cells in phospho-assays, rather we use fold-change in fluorescence or arcsinh difference in CyTOF.

Bottom line, we sometimes see results that are visually less impressive in CyTOF vs. fluorescence, but often the calculated result (#2) is still reasonable. Put another way, the typically lower background of CyTOF makes up for a certain amount of decreased sensitivity. And not everything is dimmer in CyTOF, some things are visually MORE separated in CyTOF than fluorescence, so it all depends on the antibody, label, etc. Regards,

holden

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