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Proteomic Stabilizer

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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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Jlannigan

Participant

Posts: 1

Joined: Fri Jan 23, 2015 8:39 pm

Post Mon Jan 26, 2015 5:58 pm

Proteomic Stabilizer

Hi All:
Does anyone have any experience using the SmartTube Proteomic Stabilizer for freezing whole blood (without the use of their automated base station) that was used in the recent Nolan paper "Clinical recovery from surgery correlates with single-cell immune signatures". Also does anyone have a panel of markers that they have validated for human Myeloids, particularly DCs, Basophils, and Granulocytes in whole blood. Thanks for your help!
Joanne
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mleipold

Guru

Posts: 5793

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Jan 26, 2015 6:21 pm

Re: Proteomic Stabilizer

Hi Joanne,

The HIMC has used Smart tube buffers for whole blood. I'll let my coworkers respond in more detail.

Regarding human myeloids: I'm sure some of the DC people will respond, but one place to start might be O'Gorman et al in Vaccine:

viewtopic.php?f=10&t=179


Mike
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AdeebR

Grand master

Posts: 169

Joined: Thu Mar 13, 2014 5:58 pm

Location: NYC

Post Thu Feb 05, 2015 4:40 pm

Re: Proteomic Stabilizer

Hi Joanne,

I've done some testing of the SmartTube buffer for preserving whole blood (without the base station).

It certainly seems to do a nice job of preserving the cells, and intracellular targets. Recovered frequencies of major subsets were similar before and after cryopreservation. However, the buffer seemed to degrade a number of surface antigens that are of interest to us (essentially all chemokine receptors) so we ultimately decided that it wouldn't be that useful for the study we were considering it for.

I do a lot of myeloid work with whole blood. Here's my "core" myeloid backbone for whole blood:

CD66b-152Sm
CD16-148Nd (gives strong signal on neutrophils, but never stains other subsets as well in whole blood as in PBMCs)
CD14-160Gd
CD1c-153Eu
CD141-144Nd
CD123-151Eu
HLA-DR-174Yb

CD11c-159Tb (not critical since it's expressed fairly promiscuously by human monocytes/DCs)
CCR3-164Dy (also not critical, but useful for definitively identifying eos/basophils - needs to be stained prior to fixation).

I also typically include CD19, CD3, and CD56, which are useful as exclusion parameters when identifying DCs.

Also note that whole blood is not ideal for identifying DCs on the CyTOF because you need to collect a lot of events to compensate for all the granulocytes.
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC
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flosj73

Participant

Posts: 2

Joined: Wed Jul 16, 2014 9:55 am

Post Wed Aug 26, 2015 7:11 am

Re: Proteomic Stabilizer

Hi All!

I have tried using Smart tube mainly on mouse spleenocytes and compared the signal and fold change of surface antigens and well as some intracellular phosphoproteins with 1.6% PFA fixation. After initial enhanced findings of practically everything with the proteomic stabilizer (no base instrument), I have inconsistent results with the phosphoproteins, and found that the expression of CD62L is decreased in the Smart Tube preparation when compared with PFA. I have not yet tried the chemokine receptors but after reading Abeeb's post, I won't have to.

Would appreciate hearing what others are experiencing with Smart Tube.

Florence
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prybakowska

Participant

Posts: 6

Joined: Sun Nov 06, 2016 9:56 pm

Post Mon May 08, 2017 4:27 pm

Re: Proteomic Stabilizer

HI,
I am also testing Proteomic stabilizer for the cytokine expression. In general it is doing a good job for the surface, but I am getting inconsistent results for CD16, sometimes it is preserved sometimes it is not, did someone can comments on CD16 AB in the context of proteomic stabilizer? also for TNFa-152Sm I am getting higher signal across all the populations and what is weird in the unstimulated cells compare to stimulated one and the one without fixation with proteomic stabilizer, did someone observe something like this ? any experience in cytokines preservation by proteomic stabilizer ?

Adeeb, did you switch to the BD phosflow lyse/fix buffer ? do you have any experience in cytokines preservation upon stimulation?
Best,
Paulina

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