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Mouse intra cellular staining

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Tania

Participant

Posts: 6

Joined: Wed Dec 04, 2013 12:00 pm

Post Thu Sep 10, 2015 9:49 am

Mouse intra cellular staining

Hi All,
Does anyone have an experience with staining mouse cells with intra cellular antibodies for CyTOF?
I will appreciate if you can share your experimental experience and some dot plots .

Thanks a lot,
Tania.
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Sep 10, 2015 2:56 pm

Re: Mouse intra cellular staining

Hi Tania,

To better answer your question: could you give more details?

For instance, are you wanting examples of mouse intracellular staining just as general knowledge because you plan to start those experiments? Or have you been trying to do it, and you're not having success?


-Mike
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komalkumaralienboy

Contributor

Posts: 44

Joined: Tue May 20, 2014 2:24 pm

Location: Linkoping University, Sweden and Finnish Institute of Molecular Medicine, Finalnd

Post Thu Sep 10, 2015 3:08 pm

Re: Mouse intra cellular staining

Hi Tanina,
We do Inter-cellular staining on mouse bone marrow and on spleen cells. As mike said, what do u want to know exactly?
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Tania

Participant

Posts: 6

Joined: Wed Dec 04, 2013 12:00 pm

Post Fri Sep 11, 2015 8:34 pm

Re: Mouse intra cellular staining

Hi Mike and Komal,
I'm trying to stain cells after an in-vivo stimulation with LPS, I'm using the BD commercial protocol (http://www.bdbiosciences.com/us/resourc ... tokinesfca) for the staining. However, it yields quite weak signals and minor post stimulation shifts in cytokines stainings , while I know that stimulation worked based on other markers.
So I'm looking for a protocol that will better fit my study, tips for the inhibition of cytokines secretion in in-vivo models.

Thank you for helping,
Tania
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maecker

Contributor

Posts: 23

Joined: Thu Oct 31, 2013 7:58 pm

Post Mon Sep 14, 2015 6:47 pm

Re: Mouse intra cellular staining

LPS may not be the best stimulator to start with. I would suggest PMA+ionomycin as a strong positive control to check your conditions, before moving to other less potent stimuli. Regards,

holden
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MCOlivier

Contributor

Posts: 47

Joined: Mon Oct 05, 2015 9:48 am

Location: European Genomic Institute for Diabetes, Universitity of Lille, Institut Pasteur de Lille, France

Post Tue Oct 06, 2015 11:29 am

Re: Mouse intra cellular staining

Hi Tania, Mike, Komal and Holden,

I'm frightened that there is a little misunderstanding: Tania, if I'm correct you stimulate with LPS in vivo, and then collect your cells for cytometry analysis ? If you don't see a shift either with LPS stimulation or with PMA-Iono stimulation, this culd be due to some identifiable issues:
- How long do you stimulate in vivo with LPS ? I ask because if this stimulation is too short, you may not elicit a sustained proi-nflammatory phenotype in your immune cells, so you won't see any effect of LPS stimulation vs. unstimulated state, at least regarding cytokines secretory activity, once you isolate cells and stimulate them + inhibit exocytosis. Didi you try analysis of cells from LPS treated vs untreated animals directly, without PMA-Iono + GolgiPlug ?
- I think you do, but do you stimulate cells ex-vivo with MPA-iono + GolgiPlug ? And if yes, do you do it at least for 4h in order to let cytokines accumulate in secretory cells ? Because in my hands, I was able to see cytokines without PMA-iono + brefeldinA/monencine only in some rare inflammatory PBMC in humans, and even in this case only in some inflammatory CD4+ cells regarding only some specific cytokines (which, I think, are abundant enough to be visualized without long and artificial accumulation). I have no experience with unstimulated cytokines analysis in mouse yet.

Hope it will help :geek:
Regards

Olivier

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