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Example Plot: CD69 v. HLADR on Human PBMCs?

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nlazarus

Contributor

Posts: 20

Joined: Wed Nov 13, 2013 7:28 pm

Post Mon Mar 16, 2015 11:21 pm

Example Plot: CD69 v. HLADR on Human PBMCs?

Would anyone be willing to share a representative plot of HLADR v CD69 on human PBMCs, specifically on T cell subsets? OR, some combination of activation markers that you have used to successfully pull out activated T cells? If you also have CD38 and or Ki67 included for comparison, that would be very informative

I am trying to determine the best "activation marker(s)" or combination of markers to identify subsets of activated T cells. I have been using CD38 v. Ki67 but the staining has been inconsistent with Ki67 and I am looking for a better alternative.

Thanks!

Nicole
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robs

Contributor

Posts: 40

Joined: Mon Dec 02, 2013 3:42 pm

Location: University of Connecticut

Post Wed Mar 18, 2015 3:12 pm

Re: Example Plot: CD69 v. HLADR on Human PBMCs?

Hi Nicole,
You aren't culturing are you? Because people have used IdU pulsed cells to look for proliferation with success.
Rob
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nlazarus

Contributor

Posts: 20

Joined: Wed Nov 13, 2013 7:28 pm

Post Wed Mar 18, 2015 5:53 pm

Re: Example Plot: CD69 v. HLADR on Human PBMCs?

We are not culturing the cells. After some literature searches and reviewing notes from talks I have attended, I think some combination of CD69, HLADR, CD38 and maybe CD25 may be the way to go…but if I can get away with using fewer activation markers and thus, keep additional channels "free" for other markers of interest, that is what I will do.

These are human PBMCs isolated by histopaque then frozen down…thawed and stained (without culturing or stimulating) to look at the homing receptor profile of recently activated cells in the peripheral blood 7-9 days post-natural infection (flu, respiratory, intestinal, strep, etc…). One concern is that CD69 is a very early activation marker and may not be of use at day 7(+) post-infection.

So in addition to my original question asking for a representative plot of HLADR v CD69 (vs any other "activation markers), I would like to ask for the opinion of the CyToF forum group what activation markers they typically use, and which would they recommend to comprehensively identify activation T cell subsets - memory (central, effector,etc…), T-foll. helper, Th17, Tregs, etc…


Thanks
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Mar 19, 2015 8:22 pm

Re: Example Plot: CD69 v. HLADR on Human PBMCs?

Hi Nicole,

I don't use CD69 in my panel; we just never added it. We generally consider CD38+ HLADR+ to be activated, but anything other than CD38-HLADR- (Q4) probably counts.

Regarding other T cell markers: I have attached a copy of a PBMC control. It shows some of the marker subsets you were talking about.

Generally, we consider:
CD45RA+CCR7+ = naive
CD45RA-CCR7+ = CM
CD45RA-CCR7- = EM
CD45RA+CCR7- = Eff

CXCR5+ = TFH

CCR6+CXCR3- = TH17
CCR6-CXCR3- = TH2
CCR6-CXCR3+ = TH1

CD4+CD127loCD25bright = Treg


Mike
Attachments
T cell gating-HIMC 885d4 ctrl.pdf
(366.91 KiB) Downloaded 413 times

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