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mass channel spillover

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astrid

Participant

Posts: 13

Joined: Sat Mar 25, 2017 2:20 pm

Post Thu May 11, 2017 9:52 am

mass channel spillover

Hi all

I was facing a problem in my last CyTOF run and was hoping someone could have ideas on that. I ran the same healthy blood donor PBMCs in 2 experiments 4 days apart, the cells were prepared the same way. I used the same antibody mastermix for both experiments, meaning that the mastermix was stored for 4 days in the fridge (diluted in Maxpar cell staining buffer). The samples were acquired on a CyTOF2 at a similar acquisition speed of about 400-500 events/sec. In the second run I see spillover from the 141Pr channel to some other channels it should not interact with (see attachment; no spillover to channels other than the plotted ones). Could this be due to instability of the antibodies when storing the mastermix? Does anyone have experience on storing pre-pipetted antibody mastermixes for several days?
There was an instrument wash between both runs leading to a higher Tb mean dual count, but this does not explain what I am seeing.

Any input will be welcome, thanks a lot in advance!

-Astrid
Attachments
LySt_panel 1_run1vs2_141Pr spillover.pdf
(171.39 KiB) Downloaded 291 times
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astrid

Participant

Posts: 13

Joined: Sat Mar 25, 2017 2:20 pm

Post Thu May 11, 2017 1:17 pm

Re: mass channel spillover

PS: We just looked at other data from the consecutive day (10.05.2017), having a different antibody in 141Pr and saw the same interaction between the channels, so it is not an issue of the mastermix. Any other ideas why Pr141 would interact with those other mass channels?
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ErinSimonds

Contributor

Posts: 49

Joined: Tue May 13, 2014 8:04 pm

Post Thu May 11, 2017 2:04 pm

Re: mass channel spillover

Hi Astrid,

This sounds like the Pr/Sm contamination in PFA that others have reported in the past. Rare earths are used at one point in the PFA synthesis process; in some lots they are removed and better than others.

Filtering your PFA with a 0.1 um filter or switching to a different lot (and then checking that lot!) should do the trick.

See here:
viewtopic.php?f=7&t=146

Erin
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mleipold

Guru

Posts: 2256

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu May 11, 2017 2:48 pm

Re: mass channel spillover

HI Astrid,

I would also say that in my experience, CyTOF cocktails should *not* be stored as liquid master mixes. It's an antibody-by-antibody (clone-by-clone) thing, but out of my standard panel of 33 antibodies, I have at least 6 whose background goes up by 0.5-1 log of signal across the board. This happens at *least* as rapidly as 2 days post-mix; I haven't done a formal timecourse.

Of course, that also means that 25 or so of them are fine; therefore, it's possible that your mix may be OK. But that's also something that must be tested/validated before starting to do it as a matter of course.


Mike
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GregBehbehani

Master

Posts: 80

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Thu May 11, 2017 4:03 pm

Re: mass channel spillover

Hi Astrid,

While I agree with Mike (as I usually do) about the stability of master mixes, I would point out that it appears that your machine was much more sensitive on May 9 than it was on May 5. Perhaps this antibody binding pattern (real or background) was there on 5/5 but the machine just wasn't sensitive enough to detect it. I would say that this is almost certainly the explanation for the interaction between CD8 and CD161, but for the others you're showing it's hard to say.

I have occasionally seen a small (~1%) of cells that show background staining across a wide variety of antibodies. The best explanation for this I've found was Adeeb's teams publication in which they showed this could be the result of non-specific binding of the antibodies to eosinophils and could be blocked with heparin.

The other thing to consider is that one of the interacting antibodies may have become contaminated with 141. You can easily check this by diluting them and running in solution mode. You should be able to quickly figure out the culprit.

Good luck, let us know if you sort it out.

best,

Greg
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astrid

Participant

Posts: 13

Joined: Sat Mar 25, 2017 2:20 pm

Post Mon May 22, 2017 7:22 pm

Re: mass channel spillover

Hi Erin, Mike and Greg

Thanks a lot to all of you for your input and sorry for the slow response!
I checked my PFA lot and found it to be fine (at least if I can conclude from one vial to the whole lot). Also, I checked the antibodies in question for 141 contamination and did not see anything. As mentioned, another user was observing the same interactions between those specific channels with different antibodies.
Greg, I absolutely agree that the sensitivity of the instrument is much higher on the second day and that this interaction pattern might have been there earlier!
The experiment has been done on PBMC, so eosinophils are most likely not the explanation!?
I will let you all know if I find out more!

Regards,

Astrid

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