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Low Staining on Multiple Channels

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KHLACU

Participant

Posts: 4

Joined: Thu Feb 28, 2019 2:43 am

Post Thu Feb 28, 2019 8:02 am

Low Staining on Multiple Channels

Hi all,

I have been trying to get a series of CyTOF experiments going, but in every experiment I've done some markers(out of our ~35) have been left abysmally stained. Ie max signal around 10^2. This makes it impossible to discern cell populations and start with actual samples, and not just the validations. Some of this is even occurring on DVS conjugated abs. We are using the Nolan protocols. Some staining is fine, but some is not. I am not sure what could be going wrong? Would keeping the cells at room temp for ~15 min after processing lead to Ab dissociation? What other problems could there be aside from Abs? Clearly, the protocol is being executed okay or else all the markers would be off... I don't have much CyTOF experience so any help would be great!

Thanks so much!
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KHLACU

Participant

Posts: 4

Joined: Thu Feb 28, 2019 2:43 am

Post Thu Feb 28, 2019 4:45 pm

Re: Low Staining on Multiple Channels

These cells are heavily fixed, BTW, from whole blood.
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mleipold

Guru

Posts: 2086

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Feb 28, 2019 4:51 pm

Re: Low Staining on Multiple Channels

Hi Humza,

Could you give us some example data of channels that are OK and channels that are *not* OK?

Could you give us more detail about each step of your processing? The Nolan Lab has a *lot* of protocols...........


Your comment about "heavily fixed" is one possible reason you're having low signal: not every antibody clone works properly on fixed cells.


Mike
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Hkoppejan

Participant

Posts: 3

Joined: Fri Aug 04, 2017 3:59 pm

Post Fri Mar 01, 2019 7:39 am

Re: Low Staining on Multiple Channels

Dear Humza,

You also might want to consider, eventhough Abs are purchased directly from DVS, not all clones work on fixed material.
We have experienced this for some of our antibodies and switching clones solved the problem (sometimes a particular clone works even better on fixed than fresh).
Biolegend has quite an extensive list on their website of numerous markers and clone-names and whether or not they work on fixed material.
Thermofisher has a similar list,though less extensive, in which they rate the Abs pre and post fixation.
As you mention only part of your markers show this dim signal, it's worth checking the clones you use and if alternatives are available.

List Biolegend:
https://www.biolegend.com/fixation
List Thermofisher:
https://www.thermofisher.com/nl/en/home/life-science/cell-analysis/cell-analysis-learning-center/cell-analysis-resource-library/ebioscience-resources/antibody-fixation-considerations.html

Hope this will help.

Cheers,
Hester

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