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Reagents for barcoding

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It is fine to promote your company's reagents. Just make sure they are relevant to CyTOF, and do so in moderation and style :-)
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fischerp

Participant

Posts: 1

Joined: Tue Dec 06, 2016 10:05 pm

Post Mon May 07, 2018 5:31 pm

Reagents for barcoding

Hi,
Has anyone used the eBiosciences FoxP3 Staining (fix/perm) Buffer kit for use with the Fluidigm Pd barcoding reagents? Is there any reason that this would not work?
Thanks for any assistance you can offer,
Paul
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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 Wed May 09, 2018 8:55 am

Re: Reagents for barcoding

Hi Paul,

I don't see any reason why this should not work. But the problem is that this fix/perm is much stronger than the one performed with Fluidigm fix/perm barcoding reagent, and is thus supposed to produce much more potential loss of signal for some extracellular markers (if you stain extracell after barcoding of course) as it is described by users.

To be tested ;-)

Olivier
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GregBehbehani

Master

Posts: 85

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Wed May 09, 2018 2:37 pm

Re: Reagents for barcoding

Hi Paul,

We have tested saponin perms and other perm agents with the Pd barcoding method and it should work fine; you'll probably get more barcode signal (similar to if you did barcoding after a methanol perm). The question would be if you plan to perform surface staining before or after using the eBiosciences FoxP3 Staining (fix/perm) Buffer.

If you perform barcoding and surface staining before the fix/perm step then you'll just end up needing to do some sort of fix before barcoding and the subtle barcoding perm (you'll thus effectively have two fix and two perm steps), in this case the barcoding should perform normally. If you do all of your steps after the FoxP3 Staining (fix/perm) Buffer, then your barcoding will likely give you a stronger signal, but any fixation sensitive surface markers will end up looking dimmer due to the fix/perm step. A compromise might be to stain any potentially fixation sensitive markers before fixation and barcoding, then do the fix/perm step, then barcode, then stain the remain surface markers and all intracellular markers. Which to choose depends on why your doing the barcoding and which fix sensitive antigens are important for you. Thus some pilot experiments (as Olivier suggests) would probably be a good idea.

Best of luck,

Greg

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