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Non-specific extracellular staining despite FcR blocking

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cchedid

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Posts: 4

Joined: Tue Jan 22, 2019 3:17 pm

Post Wed Jan 30, 2019 3:59 pm

Non-specific extracellular staining despite FcR blocking

Hi all,

I am new to mass cytometry and currently experimenting with a 37-marker Tcell/NKcell oriented panel on a Helios cytometer. I work with fixated, cryopreserved human PBMCs that seem normal when I look at the major cell populations in flow cytometry (mono, lympho, B cells, NK).

In mass though, after cleaning the data and gating on T lymphocytes (CD45+CD3+), I am encountering an odd non-specifically stained cell population that's always about 3% of that gate. These cells display strange/uncommon phenotypes such as CD4+CD8+, CD16+CD8+, TCRgd+CD8+... and it goes on with about 15 antibodies. And when I gate one of these populations out, all of the other strange double positives disappear.

My staining protocol goes as follows (briefly, it's the Fluidigm protocol):
- aliquot 3 million cells (in PBS)
- block FcR (using Miltenyi blocking reagent for 10min at the recommended concentration)
- extracellular staining, then 2 wash steps
- fixation/permeabilization using Fluidigm Fix I and Perm S buffers, then 2 wash steps
- intracellular staining, then 2 wash steps
- fixation with freshly prepared 1.6% FA for 10min
- wash step then overnight iridium staining.

Has anyone been encountering similar problems using the Fluidigm protocol? Unfortunately I can't seem to figure out to attach any files or images to show what this strange staining looks like.

Thanks for your response!
Cheers, Carole
PhD student at International Center for Infectiology Research (Lyon, France)
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dtelad11

Master

Posts: 107

Joined: Mon Oct 31, 2016 6:26 pm

Post Wed Jan 30, 2019 10:16 pm

Re: Non-specific extracellular staining despite FcR blocking

Naive question: have you removed doublets using a DNA versus event length gate, the Helios Gaussian paramters, or barcoding?
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AdeebR

Grand master

Posts: 169

Joined: Thu Mar 13, 2014 5:58 pm

Location: NYC

Post Thu Jan 31, 2019 1:20 am

Re: Non-specific extracellular staining despite FcR blocking

I think these staining artifacts in multiple channels usually reflect some sort of antibody/metal aggregated. Usually, filtering your antibody cocktails through a 0.1micron filter before adding to your cells solves the issue.
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC
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cchedid

Participant

Posts: 4

Joined: Tue Jan 22, 2019 3:17 pm

Post Thu Jan 31, 2019 9:30 am

Re: Non-specific extracellular staining despite FcR blocking

Hi all, many thanks for your responses.

@dtelad11, I did remove the doublets by gating on Helios Gaussian parameters vs. time, as well as 191Ir/193Ir. Wish I could find how to upload my cleaning gating strategy on here. I also performed barcoding experiments (classic Fluidigm Palladium isotopes) where I still encountered the same populations in each barcoded samples... which was surprising as I thought it would help with doublets too.

@AdeebR, good idea, I will try that ASAP! Hopefully it'll do the trick.

Thanks again!
PhD student at International Center for Infectiology Research (Lyon, France)
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ChristophS

Contributor

Posts: 21

Joined: Thu May 07, 2015 2:11 pm

Location: Switzerland

Post Thu Jan 31, 2019 10:11 am

Re: Non-specific extracellular staining despite FcR blocking

Hi Carol,

in your list of things you are doing I did not spot dead cell exclusion. Are you using CisPlatin pusling prior to the whole staining prodcedure?
Dead cells tend to attract all kinds of antibodies.

Best

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

Participant

Posts: 4

Joined: Tue Jan 22, 2019 3:17 pm

Post Thu Jan 31, 2019 12:49 pm

Re: Non-specific extracellular staining despite FcR blocking

Hi Christoph,

I did not perform CisPlatin staining as I am working with cells that were fixated immediately after extraction from the blood (using FACS lysing solution) then frozen at -80°C. So they're supposed to be all dead.

But maybe the CisPlatin staining could help discriminate between cells that were well fixated (with undamaged membranes) and cells that were poorly fixated (with damaged membranes, thus attracting all kinds of antibodies)?

However what bothers me is that this unspecific staining is not visible in flow cytometry. If it was a problem with the cells themselves, it would be apparent in flow too, right?

Thanks a ton in any case!
Cheers, Carole
PhD student at International Center for Infectiology Research (Lyon, France)
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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 Jan 31, 2019 2:28 pm

Re: Non-specific extracellular staining despite FcR blocking

Hi Carole,

What you're seeing sounds like non-specific antibody staining, that we and other have also seen. Adeeb and his group have actually published a nice paper on this in Cytometry. They showed that the effect was most likely due to cationic proteins in eosinophils and could be blocked with heparin. We have also routinely seen this effect as well. In our hands it's not clear that it is only eosinophils that cause it, but it definitely appears to be some type of mature myeloid cell, as we see a dramatically lower frequency of these events in samples from patients with AML. We have replicated Adeeb's findings that heparin will almost complete block this effect, so that would be the first thing I would try. It happens irrespective of barcoding. I don't have the reference for Adeeb's paper handy, but perhaps he can chime in with it, (it is on Cytoforum).

Antibody aggregates are also a possibility, but in our experience these tend to only have a signal on a single channel. Also, unless these are also somehow stuck to your cells, they won't get debarcoded into a cell population by the debarcoding software. It is still a good idea, as Adeeb suggests, to filer your staining cocktail through a 0.1um filter, or to spin it at high G to pellet out any antibody aggregates.

I'm not sure what you mean by "And when I gate one of these populations out, all of the other strange double positives disappear." Perhaps you could tell us more about this?

I was curious about one thing, how did you do the barcoding without first fixing the cells? In our experience (and that of others) the cells tend to die if you do the partial permeabilization on live cells.

best of luck,

Greg
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ChristophS

Contributor

Posts: 21

Joined: Thu May 07, 2015 2:11 pm

Location: Switzerland

Post Thu Jan 31, 2019 3:41 pm

Re: Non-specific extracellular staining despite FcR blocking

Hi Carole,

in this case with already fixed cells I am not sure if CisPt would discriminate much anymore.
I would try the Heparin method suggested and on the other hand you say that gating out obviously implausible cells (e.g. CD4+CD8+ in PBMC) takes care of all unexpected signals. This sounds like a workaround already like gating out the diagonal in a fluorscence experiment when no DCE was used (or when PI is used).

Best

Christoph
(the french tend to give me an "e" in the end, but now I stole it from you in the first post; desolé)
Christoph Schwärzler
Director Cytometry
Flow Cytometry Facility (https://www.cytometry.uzh.ch/en.html)
University Zürich
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mleipold

Guru

Posts: 2095

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Jan 31, 2019 3:47 pm

Re: Non-specific extracellular staining despite FcR blocking

Hi Carole,

You should be able to attach files, like to illustrate your gating strategy.

Immediately under this posting window, there are two tabs on the left: Options, and Upload Attachment.

Upload Attachment allows you to upload PDF, PPT, Excel, etc. This can be "in-line" so the picture is positioned in your post where it's relevant, or can simply be attached as a file link at the end of the post. There *is* some upper limit to the file size. I can't remember what, at the moment, but I recall that it does give you an error message (something like "File exceeds X limit").

If you are still not able to attach a file, please let us know and we'll try to see what's affecting your account.


Mike
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mleipold

Guru

Posts: 2095

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Jan 31, 2019 7:14 pm

Re: Non-specific extracellular staining despite FcR blocking

Hi Carole,

1. What concentration are you running your cells at? Yes, debarcoding and other methods do usually get rid of doublets, but you can imagine that if you have a *lot* of them, then a larger fraction might slip through, especially depending on your debarcoding parameters.

For reference: the HIMC generally runs cryopreserved PBMCs at 0.8M/mL (~250 events/sec), and whole blood samples (SmartTube, etc) at ~0.6M/mL (~180 events/sec). We find that these are good rates to minimize doublets and avoiding clogs (particularly if using the Helios PSI), while still not taking *forever* to run.


2. El-ad also suggested Ir vs Event Length. You responded "@dtelad11, I did remove the doublets by gating on Helios Gaussian parameters vs. time, as well as 191Ir/193Ir." Have you taken a look at Ir vs EL?

3. Greg mentioned Adeeb's heparin block paper. Here's the link: https://doi.org/10.1002/cyto.a.22826



Mike
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