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different metal channels, same mass

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Wkennedy

Participant

Posts: 5

Joined: Fri Dec 11, 2015 10:23 pm

Post Sat Oct 08, 2016 12:32 am

different metal channels, same mass

Hi guys,

Three questions about my run!

When running CyTOF acquisition, we had left two of the same mass channels open (CE142 and Nd142). When I'm looking at the data, I was wondering if there is increased potential for spillover into the +1 and +16 channels when two masses of the same weight are open?

Second, When looking at my nuclear stain, I think I am using too much, but I just wanted to clarify. If there isn't excess staining (I still see clean cell events), is there a problem that it is this high?

Third: I have been seeing this "wandering" population that I am seeing in IgD+, CD31+ in the image attached is an issue of panel design?

Thanks in advance for all your help!
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Cytobank question oct 2016.png
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Oct 10, 2016 2:50 pm

Re: different metal channels, same mass

Hi,

I'm not sure what you mean by "increased potential for spillover into the +1 and +16 channels when two masses of the same weight are open". The CyTOF doesn't "know" any difference between Ce142 and Nd142. Remember, in CyTOF, mass is actually time-of-flight: all the CyTOF "knows" is that ions are striking the detector at a particular time interval. That time interval has been calibrated during machine-building and during tuning so that the software interprets that as being mass 142 (it can't distinguish between two elements of the same mass).

Regarding your Ir staining: I think it looks fine. I typically have my Ir staining around 1e3 as well: it does a good job of isolating the Ir+ cells away from debris. I've seen right-leg-spillover issues into the 194 channel if my Ir staining gets to 1e4, though.


Mike
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AdeebR

Grand master

Posts: 169

Joined: Thu Mar 13, 2014 5:58 pm

Location: NYC

Post Mon Oct 10, 2016 2:56 pm

Re: different metal channels, same mass

I believe spillover into the +1 and +16 channels is a function of how much of the base isotope is present in the sample and it's oxidation potential, regardless of whether or not you are recording data for that channel. i.e., I would expect that the amount of spillover that you detect the Gd156 channel would not be affected by whether or not you record the Ce142 and Nd142 channels. The fact that you have two distinct isotopes contributing potential spillover could have an effect, but since Ce142 is a relatively minor isotope of cerium (~11%) and this element is not generally used in antibody staining, I would imagine that this contribution shouldn't be too problematic.

Your nuclear stain is perhaps a little high but doesn't seem to be too bad. The biggest concern with high nuclear stain is the increased potential for "streaking" and ion cloud fusion, so so as long as you are seeing clean separation of events it shouldn't be a problem.

The wandering population is likely an artifact related to non-specifically staining antibody or metal aggregates. Generally, I've found that filtering your antibody cocktail through a 0.1micron filter helps to minimize these artifacts.

Adeeb
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC

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