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vrozot

Participant

Posts: 13

Joined: Fri Jan 31, 2014 12:52 pm

Post Thu Apr 20, 2017 2:22 pm

Re: background

Hi Astrid,

Could it be Barium contamination?
When you look your Ir vs Ba channels do you see they colocalize?
I have already seen an heavy barium contamination spreading quite far in the spectrum.
Just an idea.

Best,

Virginie
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astrid

Participant

Posts: 13

Joined: Sat Mar 25, 2017 2:20 pm

Post Thu Apr 20, 2017 4:26 pm

Re: background

Hi all,

thanks a lot for your inputs! No, it is not Barium, I have checked for that. I will test my PFA and all my buffers and keep you posted!

Kind regards,
Astrid
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ekiiadiri

Participant

Posts: 1

Joined: Mon Oct 24, 2016 7:18 am

Post Fri Apr 21, 2017 6:30 am

Re: background

I think you should check channel 138. Your buffer likely contains Ba 138 and this could be the source of the contamination in the La 139 channel especially if the barium concentration in the buffer and the speed of acquisition are high.
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astrid

Participant

Posts: 13

Joined: Sat Mar 25, 2017 2:20 pm

Post Mon Apr 24, 2017 10:36 am

Re: background

Hi all

I wanted to follow up on our discussion: I ran all my buffers as well as PBMCs treated with different fixatives on our instrument and I indeed observed this strong background signal in my staining buffer, which is PBS+1%BSA (the PBS alone was fine). The signal is strongest around the 139-142 channels but is visible up until 170. So it seems there is a lanthanide contamination in the BSA, most likely La139 (or a poly-contamination). Has anyone experienced this? The PFA was fine...

Astrid
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astrid

Participant

Posts: 13

Joined: Sat Mar 25, 2017 2:20 pm

Post Mon Apr 24, 2017 6:20 pm

Re: background

No, it is not Barium, the signal is visible in the 138 channel but stronger in the 139-142 channels. See attachment (it's a run on buffer only --> PBS+1%BSA, no cells).
Attachments
17-04-24_PBS-BSA.pdf
(30.72 KiB) Downloaded 353 times
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ChristophS

Contributor

Posts: 21

Joined: Thu May 07, 2015 2:11 pm

Location: Switzerland

Post Wed Apr 26, 2017 8:35 am

Re: background

Hi Astrid,

since no single element would have an isotope composition that could explain this pattern it looks a bit like the BSA you use may have traces of Mischmetal (https://en.wikipedia.org/wiki/Mischmetal) which typically is composed of a majority of Cerium (you should see ~8% of your 140 signal in 142) and other lanthanides. It is used in lighter flints and (wild hand-waving) the cattle may have ingested one of those or the pastures are in the vicinity of mines or production plants.

Hope this may help to shed light on the problem. BTW did you check alternative sources of BSA?

Christoph
Christoph Schwärzler
Director Cytometry
Flow Cytometry Facility (https://www.cytometry.uzh.ch/en.html)
University Zürich
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astrid

Participant

Posts: 13

Joined: Sat Mar 25, 2017 2:20 pm

Post Thu Apr 27, 2017 3:58 pm

Re: background

Hi Christoph

Thank you very much for this input, this explanation in deed seems to match the pattern I see...!
It seems to be LOT-specific since the last one (ordered more than a year earlier than the present one) did not show any of that background signal. I will test BSA in the future before using...
Thanks again!

-Astrid
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Apr 27, 2017 4:08 pm

Re: background

Hi Astrid,

Do you still have any of the "bad" BSA lot? If so, could you try filtering it (0.1um filter) and running some of it in Liquid mode again? It would be useful knowledge for the community if you found whether (or not) you could remove the particulates by filtration. If you can, great.

If you can't, then either the particulates are too small for the filter to trap, or they're dissolved metal ions in solution (in either case, the BSA lot would be unusable for CyTOF).


Mike
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astrid

Participant

Posts: 13

Joined: Sat Mar 25, 2017 2:20 pm

Post Mon May 01, 2017 7:46 pm

Re: background

Hi Mike,

yes, I still have some BSA of that lot, will try filtering it and get back to you!

Astrid
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astrid

Participant

Posts: 13

Joined: Sat Mar 25, 2017 2:20 pm

Post Mon May 22, 2017 7:06 pm

Re: background

So, I finally found the time to run the BSA again. Filtering (using a 0.22 um filter I had at hand) did not change anything, the background signal was still visible to the same extent!

-Astrid
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