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Fluidigm anti human TCR gd

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xiaoxiaoUC

Contributor

Posts: 20

Joined: Sun Feb 04, 2018 4:48 pm

Post Tue Feb 13, 2018 7:19 pm

Fluidigm anti human TCR gd

Hi guys,

In my panel, I have anti human TCR gamma and delta antibody from Fluidigm (clone: 11F2). Do you know does this antibody target on some specific TCR V gamma chain or V delta chain? I checked some references that used this antibody. I didn't find any information about the chain target. It looked like this is for general gamma and delta T cells, am I right?

Thank you!

Xiao
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jonfisher

Participant

Posts: 3

Joined: Tue Feb 07, 2017 3:54 pm

Post Tue Feb 13, 2018 7:51 pm

Re: Fluidigm anti human TCR gd

Hi Xiao,

This clone (11F2) apparently binds to the framework region of the gdTCR (according to Fisher Scienfic https://www.fishersci.com/shop/products ... /bdb347903)

To be honest, we have found that many of the "pan-gdTCR" antibodies have variable affinities for different gdTCR subtypes. The Biolegend clone B1 works well in our hands, and you could just conjugate it yourself if you're concerned about catching every last one gdT cell in your sample. Beware, if you're planning on *also* staining for a particular Vg or Vd chain, many of the pan-gdTCR antibodies interfere with binding of Vg or Vd specific antibodies.

I hope this helps

BW

Jon
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xiaoxiaoUC

Contributor

Posts: 20

Joined: Sun Feb 04, 2018 4:48 pm

Post Tue Feb 13, 2018 8:00 pm

Re: Fluidigm anti human TCR gd

Hi Jon

Super helpful! Thanks a lot.

Xiao
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AnnaBelkina

Contributor

Posts: 23

Joined: Sat Nov 01, 2014 7:07 pm

Post Tue Feb 13, 2018 8:40 pm

Re: Fluidigm anti human TCR gd

Hi Xiao,
I haven't used the mass cytometry ab but worked with this clone a lot in flow. It is described as a pan-gamma-delta antibody but there are several publications that describe it only staining Vdelta1+ cells. It appears that it binds way better to Vdelta1 then Vdelta2 TCR, so often the Vdelta2 gdT cells are too "dim" and are indistinguishable from gdTCR negative cells. It is also quite finicky when combined with CD3 staining with a bunch of aCD3 clones since it looks like their target epitopes are too closely situated in the TCR complex. To get best results, I recommend adding the 11F2 to the cells first, incubate for 5 min, then add the rest of the antibodies. But again, this is flow cytometry experience.
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xiaoxiaoUC

Contributor

Posts: 20

Joined: Sun Feb 04, 2018 4:48 pm

Post Wed Feb 14, 2018 4:02 pm

Re: Fluidigm anti human TCR gd

Anna

Nice point! I also noticed this antibody has different affinity with different gd T-cell subpopulations. Some patients data showed the competition with CD3 staining like what you mentioned because the antibody targeting part of TCR closely to CD3. However, some patients data had a nice staining with both of them, which may indicate the TCR structure difference and the geometry difference between TCR and CD3 in these patients.

Thanks for sharing your experience.

Xiao
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jonfisher

Participant

Posts: 3

Joined: Tue Feb 07, 2017 3:54 pm

Post Wed Feb 14, 2018 4:36 pm

Re: Fluidigm anti human TCR gd

Hi Xiao,

Anna is correct - we tried to get around the same issue by staining in different orders as she describes, and it does work.

What type of samples are you staining? If you're taking samples from a tumour or epithelial compartment, then a pan-gdTCR antibody with subsequent subtyping is probably needed as the non-Vd1/Vd2 subsets are generally tissue-resident. However, if you're dealing with peripheral blood samples, you could just stain for Vd1 and Vd2, there are loads of antibodies which discriminate these really nicely, and non-Vd1/Vd2 gdT cells are much rarer there. There's more information on this here:
http://clincancerres.aacrjournals.org/content/20/22/5720?ijkey=c32ac9aca72f14f73613a6627320ce602e0487af&keytype2=tf_ipsecsha

You mentioned:
some patients data had a nice staining with both of them, which may indicate the TCR structure difference and the geometry difference between TCR and CD3 in these patients.


I suspect that this may be to do with the varying affinity of the pan-gdTCR antibody with different gdT cell subsets, rather than geometric alterations within one subset. For example, patients who are CMV+ are thought to have higher Vd1 proportions, as are patients from malaria endemic areas - a higher Vd1 proportion, considered alongside the staining issues that Anna mentioned, would give "better" staining in Vd1-hi donors. Conversely, CMV- Western Europeans living in Western Europe typically have higher Vd2+ proportions, which may give you poorer staining. There's quite an interesting literature on this, but in summary it's probably due to different innate immune responses against pathogen-associated molecular patterns.

BW

Jon
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cguidos

Contributor

Posts: 28

Joined: Tue Nov 18, 2014 3:10 am

Post Wed Feb 14, 2018 5:35 pm

Re: Fluidigm anti human TCR gd

Hi all

We have not used Fluidgim's 11F2 TCRgd Ab (which looks very dull on their TDS sheet), but have used B1 and and 5A6.E9. The latter stains 1-2 orders of magnitude brighter than B1 or 11F2. We purchased the 5A6.E9 hybridoma, purified the Ab and tagged it ourselves - it works very nicely!

Cindy
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mescorcio

Participant

Posts: 1

Joined: Mon Sep 19, 2016 3:24 pm

Post Thu Feb 15, 2018 9:24 am

Re: Fluidigm anti human TCR gd

Hi Xiao,

We are looking at peripheral blood cells and chose to conjugate anti-Vdelta1 and anti-Vdelta2 antibodies ourselves because of poor results with the pan-gd TCR in flow. Also, we are particularly interested in the Vd2 population and variations in its subsets across different donors. You can see more about how we type them here: http://www.pnas.org/content/113/50/14378

Best wishes,

Monica.

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