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High CD66+ Population

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hchen95

Participant

Posts: 3

Joined: Fri Feb 08, 2019 10:59 pm

Post Sat Feb 09, 2019 12:35 am

High CD66+ Population

Hey all,

When we were analyzing our CyTOF samples, we noticed this extremely high CD66+ population, and we were wondering if anyone else has encountered or experienced something like this?
We're currently using a CD66 clone conjugated to Metal 140 and staining on SmartTube fixed whole blood.

Image

Any thoughts and ideas would be appreciated, thanks!
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mleipold

Guru

Posts: 2086

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Feb 11, 2019 5:12 pm

Re: High CD66+ Population

Hi Han,

Could you give us your gating strategy, starting from Ungated cells (ie, hot off the CyTOF)?

Since you're using channel 140 for CD66, one potential explanation for your CD66hi cells is that they're "EQ Bead+Cell" doublets that haven't been removed.


Mike
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hchen95

Participant

Posts: 3

Joined: Fri Feb 08, 2019 10:59 pm

Post Mon Feb 11, 2019 6:18 pm

Re: High CD66+ Population

Hey Mike,

Here is a quick schematic of our gating hierarchy.
Image

Starting from our ungated population
1) we first plots channel 142 vs channel 151 and gate out the double positive cells
2) plot channel 165 vs channel 175 and gate out any remaining double positive cells (as you can see, it tends to be pretty clean the first time around)
3) Gate DNA vs CD235a to exclude RBCs and doublets
4) Gate CD66 vs CD45 to for lymphocytes/granulocytes.
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nkhanbham

Master

Posts: 53

Joined: Wed Feb 25, 2015 3:03 pm

Post Mon Feb 11, 2019 6:33 pm

Re: High CD66+ Population

Hi Han Chen,
We use the CD66b-152Sm conjugate from Fluidigm and it is also very bright like yours (>2000 ion counts). To minimize spillover into the nearby and +16 channels we titrated it down so ion counts were just under 1000. You could try doing the same with your 140Ce conjugate if spillover is a problem.
NK
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henkmei

Participant

Posts: 16

Joined: Wed Jul 30, 2014 9:44 pm

Post Mon Feb 11, 2019 9:36 pm

Re: High CD66+ Population

Hi Han,

do you use heparin in your protocol to prevent artefactual staining of eosinophils?

https://doi.org/10.1002/cyto.a.22826

Henrik
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hchen95

Participant

Posts: 3

Joined: Fri Feb 08, 2019 10:59 pm

Post Fri Feb 15, 2019 10:28 pm

Re: High CD66+ Population

Hi NK,

We're not having a huge problem with spillover, rather we're primarily curious as to what might be causing this population.

Henrik - we do not use heparin in our protocol and that's an interesting suggestion. We'll have to look into it further.

Thanks!
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AndrewD

Participant

Posts: 9

Joined: Tue Feb 20, 2018 7:18 pm

Post Thu May 16, 2019 12:28 pm

Re: High CD66+ Population

Hi Han,

The CD66b+ surface phenotype in PBMC are most likely low density granulocytes. https://link.springer.com/article/10.1007%2Fs00281-013-0375-7.

These cells should also be CD16bright and CD10+.

We also see these cells in our PBMC samples to varying percentages which increase in autoimmunity as described in the linked paper.

HTH
Andrew

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