FAQ  •  Register  •  Login

Combining surface staining with phospho

<<

Kjwaller

Participant

Posts: 11

Joined: Sat Aug 27, 2016 6:56 am

Post Mon Sep 05, 2016 11:11 am

Combining surface staining with phospho

Hi,

I have been trying to optimise my stimulations etc. for phospho flow using "normal" flow cytometry before I move over to my cytof panel and am having a few issues with the order. If I surface stain after fixing I tend to lose some of my surface markers, this is also true after stimulation. However if I stain my surface markers first my CD14 blocks my subsequent LPS stimulation. Does anybody have any advice for the best sequence when it comes to surface staining and phospho staining? It may be that it is more successful with cytof, I don't know yet. On a slightly separate note, does anyone have experience with phospho flow for stat2?

Thanks,
Kate
<<

RuthCyTOF

Participant

Posts: 5

Joined: Sat Nov 30, 2013 10:19 pm

Post Wed Sep 07, 2016 10:42 pm

Re: Combining surface staining with phospho

Hi Kate
For our studies to measure TLR4 levels we had a similar issue with CD14 labeling. We have identified monocytes with CD4 which shows dim staining. They are easily distinguishable from CD4hi T cells and you can verify this by testing with CD11c and CD14 to see how the populations overlap.
Ruth
<<

GregBehbehani

Master

Posts: 78

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Thu Sep 08, 2016 12:10 am

Re: Combining surface staining with phospho

I typically stim and then fix (with PFA only). Many stims peak after a 10-15 minutes, so you really need to fix just after the stim to "freeze" the signaling. If you add antibodies to the cells first I think you could introduce a whole host of artifacts. If you use a relatively low PFA concentration for your fix, most surface antigens still look pretty good. If you're having trouble with a few in particular, you may want to just try a different antibody clone.

Good luck,

Greg
<<

komal2000

Participant

Posts: 2

Joined: Mon Aug 29, 2016 12:55 pm

Location: University of Helsinki, Finland

Post Thu Sep 08, 2016 7:15 am

Re: Combining surface staining with phospho

Hej,

I had the same problem before
U could try this
1. Stain, Stimulate, fix with FA, perm and stain for intercellular
2. Stimulate, Fix with FA, stain, perm and stain for intercellular
3. Stimulate, Stain, fix with FA, perm and stain for intercellular.

and also think about the fluorochromes some of them are very sensitive for fix and perm buffers.

What is your fix and perm buffers?

Sincerely
Komal
<<

mleipold

Guru

Posts: 1778

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Sep 08, 2016 7:02 pm

Re: Combining surface staining with phospho

Hi Kate,

As Greg was saying, several clones are more sensitive than others to fixation. For example, as far as I know, the B73.1 clone for CD16 is generally considered to be the best post-fix clone...3G8 clone is often screwed up by fixation.

If you haven't already, I would recommend checking other CyTOF phosphoflow papers against your clone list to confirm that you're using the best clones. That said, there often is a decrease in signal post-fix, even with the "good" clones. Similarly, the perm'ing agent that you use can make a different as well: saponin usually doesn't screw up surface markers, but methanol sometimes does. However, you may have to live with it, as methanol is usually required for STATs, among others.


mike
<<

AdeebR

Grand master

Posts: 167

Joined: Thu Mar 13, 2014 5:58 pm

Location: NYC

Post Fri Sep 09, 2016 2:23 pm

Re: Combining surface staining with phospho

Hi Kate,

As several others have already suggested, our typical preferred workflow for phospho signaling is to stimulate, then do a "mild" fix with 1% formaldehyde for 10 mins, surface stain, and then proceed to methanol permeabilization before staining for the intracellular phospho proteins. Using this protocol we typically have no issues resolving most major immune subsets (CD14+ expression using clone M5E2 is usually easy to resolve).

However, there certainly are some antibodies where do do lose staining even with mild fixation, and in these cases the only option we've found is to add the antibodies to the cells prior to fixation. While you observed that some antibodies can block stimulation, we've also come across situations in which antibodies can induce signaling, or modulate signaling pathways, which can confound your results. The effects can sometime be subtle, so whenever we take this approach we always do pilot experiments where we test our whole signaling panel and stimulation protocol with and without the addition of the staining antibodies to determine whether any of the pathways are being affected.

This can be pretty laborious and expensive, so we typically only go through with this only if determining expression of the fixation sensitive protein in direct conjunction with signaling profiling is absolutely critical for an experiment (in many experimental setups it isn't). If it's not, it's generally much easier to just run parallel aliquots of the samples (i.e., one where we include the surface markers on the cells prior to fixation, and another where we look at signaling), and include a sufficiently large panel of fixation resistant surface markers to be able to identify the analogous populations in both samples where you want to compare protein expression.

Adeeb
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC
<<

Kjwaller

Participant

Posts: 11

Joined: Sat Aug 27, 2016 6:56 am

Post Wed Sep 28, 2016 2:30 pm

Re: Combining surface staining with phospho

Thank you all for your answers, I have been trying to optimise with flow cytometry using the sequence: stimulate, fix (BD fixative), surface stain, perm (BD perm III) then intracellular stain, my results have been hit and miss, sometimes it's only a slight drop in signal and sometimes nothing. I have also attempted stimulate, cold PBS wash (to stop signalling), surface stain on ice, fix and perm then surface stain, I found my surface stain to be ok but my staining for STATs was decreased, I don't think it stopped the reaction as well. I have the fluidigm fix and perm buffer (all in one) but haven't tried it out yet. Has anyone got experience with this buffer? Is it any better or should I use 1% PFA and methanol instead?
Thanks in advance,
Kate

Return to Introduction to mass cytometry

Who is online

Users browsing this forum: No registered users and 2 guests