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Combining Phospho and Cytokine Staining

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garguelles

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Joined: Thu Jun 16, 2016 10:52 pm

Post Fri Jun 17, 2016 5:32 pm

Combining Phospho and Cytokine Staining

Hello,

I am attempting to design a panel combining surface, cytokine, and phospo staining for CyTOF. I thought it was possible to combine cytokine and phospho staining in one panel, but that the permeabilization steps may need to be altered. I haven't been able to find a protocol for this.

Does anyone have any experience with this?

Thanks.

Gabriel
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mleipold

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Location: Stanford HIMC, CA, USA

Post Fri Jun 17, 2016 7:05 pm

Re: Combining Phospho and Cytokine Staining

Hi Gabriel,

Phospho and cytokine staining are not usually done in the same tube, even if you're looking at basal levels (rather than stimulated).

1. You need to fix cells in order to stop the phosphatase activity that would decrease your phospho signals. However, most ICS protocols and a lot of surface phenotyping protocols are written for staining *live*, unfixed cells.

2. ICS is most often done using more gentle perms like saponin, rather than methanol. Methanol is a harsh perm which usually does not preserve 3D epitopes. Antibodies *can* be raised against linear epitopes, but those aren't the most common ICS ones.


So, even if you're just looking at basal levels, you'd have to hunt for both surface and cytokine antibodies that can withstand both fixation and methanol.


If you're stimulating cryopreserved PBMCs, I don't think you can really do it because of stimulation timelines: phospho stims are usually for 15min or so, whereas ICS stims are for 4-6 hr (or overnight), depending on the stim. Phosphos are usually transcription factors that get phosphorylated to direct a cell to make a particular protein/cytokine. So, signalling happens quickly and then turns off quickly, whereas it can take a few hours (even with brefeldin) to build up enough cytokine levels to detect. See Frei et al, ( http://dx.doi.org/10.1038/nmeth.3742 ) Fig 1D for an example of the timing difference between mRNA synthesis and protein levels (and phospho signalling technically would happen upstream of the mRNA synthesis).


Mike
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hluche

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Post Sun Jun 19, 2016 9:42 pm

Re: Combining Phospho and Cytokine Staining

Hi all,

To add on the answer of Mike, there is to my knowledge two papers trying to combine pFlow and ICS detection by conventional flow.
1) http://onlinelibrary.wiley.com/doi/10.1 ... 22444/full
In this (very nice) paper, they worked on finding the optimal fix/perm buffer condition to simultaneously detect pErk that is supposedly a pretty much stable pModification together with Ifng and Perforin.
2) http://dx.doi.org/10.1155/2014/671431
In this paper, they did p38/MAPK, IFNg and IL10 on PBMCs and also optimised staining procedures. Key there was again that this MAPK phosphorylation are very stable over PMA/Iono stimulation.
This is also the same pModification that is revealed together with RNAs in the paper from (Frei et al., Nat Meth, 2016).

The relevance of such combined detection of pflow and ICS will very much depend on the pModification you are after as both pFlow and ICS are not following the same kinetics upon cell activation as mentioned by Mike.

Hervé

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