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Staining nuclear transcription factors for T helper subsets

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dmostag1

Participant

Posts: 3

Joined: Mon Sep 10, 2018 1:49 pm

Post Mon Sep 10, 2018 4:53 pm

Staining nuclear transcription factors for T helper subsets

Hello Forum,

I am curious whether anyone has been able to gate T helper subsets during phospho-CyTOF. Our protocol uses a 2% PFA fixation before surface staining, then methanol permeabilization before intracellular staining. Unfortunately, the surface markers CCR6 and CXCR3 are incompatible with fixation before staining. I was mainly wondering whether anyone has tried using any clones for the canonical T helper subsets (ie T-bet, Gata-3, and Ror-gt) to gate these subsets, how successful they were, or if there is a better solution.

Any help is very much appreciated,
Darius
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Verhoeff

Participant

Posts: 19

Joined: Wed Mar 02, 2016 10:25 am

Post Tue Sep 11, 2018 8:26 am

Re: Staining nuclear transcription factors for T helper subs

Dear Darius,

In our lab we include an additional staining step before fixation, with the markers that are affected by fixation. We barcode samples afterwards and stain the other markers. This means that there is a slight technical variability between samples for the CCR6 and CXCR3 so we don't use them as input for clustering algorithms. However signal intensities can be evaluated, with a slightly higher range between samples.


Cheers,
Jan
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dmostag1

Participant

Posts: 3

Joined: Mon Sep 10, 2018 1:49 pm

Post Tue Sep 11, 2018 1:44 pm

Re: Staining nuclear transcription factors for T helper subs

Hi Jan,
Unfortunately for our phospho-cytof, we stimulate our samples for a very short, fixed amount of time before we have to fix the cells to quickly and suddenly capture a snapshot of cellular activities. I'm worried that by performing a surface stain before fixation that we prolong sample stimulation and cell signaling and also introduce potentially confounding cell signaling.
Thanks for the help,
Darius

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