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Staining for cytoplasmic and nuclear antigens

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SaraAli

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Joined: Wed Jan 10, 2018 3:37 pm

Post Wed Jan 24, 2018 5:08 pm

Staining for cytoplasmic and nuclear antigens

Hi,

My panel consists of cell suface, cytoplasmic and nuclear markers, and I was wondering if there's a way of staining them all simultaneously using Maxpar reagents. The Maxpar staining protocols explain how to stain for either cell surface and cytoplasmic antigens or cell surface and nuclear antigens but I want to stain them all at the same time.

Once again, many thanks for this incredibly helpful forum!
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mleipold

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Location: Stanford HIMC, CA, USA

Post Wed Jan 24, 2018 7:03 pm

Re: Staining for cytoplasmic and nuclear antigens

Hi Sara,

At least in theory, you could do that.

The trouble comes when a step is affected by another step. For example, many of the intracellular cytokine assays are normally done with surface staining while the cells are still alive (ie, pre-fixation). As such, some of the surface clones might be affected by fixation prior to staining.

Similarly, some surface antibodies are ok with fix, but are affected by methanol-perm prior to staining. In other cases, cytoplasmic might be OK with something like saponin, but could be affected by the special nuclear buffers.


The only way to really find out is to do the experiment where you compare all the combinations of staining, fix, and perm step orders.


Mike
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sfauteux

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Post Wed Jan 24, 2018 7:43 pm

Re: Staining for cytoplasmic and nuclear antigens

Hi Sara,

Without any specification on the panel I would answer that some epitopes survive methanol, other don't.

Perhaps this is something you could test (I remember at some point fluidigm offering 25 tests tubes, but they are hard on the negociating part...)

In our hands, traditional fixation/permeabilisation works better than Maxpar, but that may be only a locally biased effect. By traditional I mean: diluting saponin, methanol and PFA in your own buffers.

Best of lucks, CyTOF takes a lot of time to set up but works nice once this is done, if no stability caveats :p
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GregBehbehani

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Location: The Ohio State University, Columbus, Ohio

Post Wed Jan 24, 2018 11:54 pm

Re: Staining for cytoplasmic and nuclear antigens

Hi Sara,

I would echo Mike's comments; it's certainly possible to do this, but it will all depend on the specific antigens and antibodies you're studying.

Most of the original phospho-flow studies done in the Nolan lab were performed with a single post-methanol staining step, but this greatly limited the surface markers that could be studied to those that were stable after methanol (in my experience, only about 50% of biologically relevant surface proteins). If you're perm is saponin, then you'll fare much better (most surface proteins are fine after saponin), but just be aware of the recall that Fluidigm had on some of their buffers, as a small subset of Fluidigm antibodies are labeled with a different polymer (other than X8) which can non-specifically bind to some lots of saponin. You should also be aware that some surface proteins have an intracellular pool that can cause post-perm staining to be brighter than pre-perm (and potentially change immunophenotype assignments).

Fortunately, if you've already got a protocol that works by regular flow with a single staining step, then you should be fine to just convert this to a CyTOF experiment (same fix/perm, just substitute metal labeled versions of the same antibody clones you use). Otherwise, as Mike suggests, you'll have to validate the optimal conditions under which your antibodies bind (if this isn't already published). Fortunately, without having to worry too much about compensation, you can quickly test your whole panel under a variety of fix/perm conditions and you should be able to sort this out fairly quickly.

Best of luck,

Greg
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SaraAli

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Joined: Wed Jan 10, 2018 3:37 pm

Post Thu Jan 25, 2018 5:09 pm

Re: Staining for cytoplasmic and nuclear antigens

Thanks, Mike and Sebastien, for your suggestions. Much appreciated.

Greg - Thanks for your comment too. I was just reading your paper on using partial permeabilisation with saponin for barcoding. The bone marrow aspirate samples used in the paper were freshly isolated and immediately fixed with a special kind of buffer. I was wondering if you had tried the same protocol with bone marrow aspirate samples that were cryopreserved in the traditional way and whether the results were comparable?
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GregBehbehani

Master

Posts: 85

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Thu Jan 25, 2018 6:06 pm

Re: Staining for cytoplasmic and nuclear antigens

Hi Sara,

You're very welcome; we're all happy to help!

As for your question about pre-perm barcoding: Yes, it works fine on Ficolled cryopreserved cells, but we still fix these before the pre-perm step as we have always assumed that the live cells would not like to have their membranes partially permeabilized (though we never really tested that; if kept cold, perhaps they could tolerate it). You can barcode live cells without the fixation or the pre-perm, but this requires much larger amounts of barcode and (as essentially all barcoding reagents also function as live-dead stains) any dead cells will have a very high level of barcode signal.

An alternative approach published by Mike's group in the Stanford HIMC is to use barcoded CD45 antibodies, and this also works well, but is a bit more complicated and doesn't allow for barcoding of CD45 negative cells.

best of luck,

Greg
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jonfisher

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Joined: Tue Feb 07, 2017 3:54 pm

Post Thu Jan 25, 2018 10:18 pm

Re: Staining for cytoplasmic and nuclear antigens

Hi Sara,

I've got protocols that work for surface then cytoplasmic staining - I use the MAXPAR fixation buffers, then do a light perm for barcoding before washing off the perm buffer and resuspending in PBS, then I stain the surface and perm again with methanol for cytoplasmic staining. Whilst some of the staining isn't as bright as it would be on unfixed cells I still get good signal.

I'm away from London at the moment but I can hook you up with someone in our lab at UCL-ICH who's done the protocol with me. I figure it's just up the road so might help.

BW

Jon
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SaraAli

Participant

Posts: 10

Joined: Wed Jan 10, 2018 3:37 pm

Post Fri Jan 26, 2018 12:03 pm

Re: Staining for cytoplasmic and nuclear antigens

Hi Jon,

That would be really great! Thanks. Will PM you.
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AdeebR

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Location: NYC

Post Fri Jan 26, 2018 3:26 pm

Re: Staining for cytoplasmic and nuclear antigens

GregBehbehani wrote:Hi Sara,
...we have always assumed that the live cells would not like to have their membranes partially permeabilized (though we never really tested that; if kept cold, perhaps they could tolerate it)...
Greg


I think that's a solid assumption. I ran a couple of experiments a few years ago to see if we could perform a mild permeabilization on non fixed cells but even very low doses of saponin were not tolerated by live cells.
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC
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GregBehbehani

Master

Posts: 85

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Fri Jan 26, 2018 4:54 pm

Re: Staining for cytoplasmic and nuclear antigens

Thanks Adeeb! I always wondered, but it's importantly to actually do the experiment.

best,

Greg
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