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Odd double positive staining

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ndawson

Participant

Posts: 13

Joined: Wed Jan 08, 2014 4:40 pm

Post Tue Mar 24, 2015 12:54 am

Odd double positive staining

I see this double positive 45 degree population every so often and I'm not quite sure what it is. Before, I have been able to gate it out using my cisplatin gate but this population is clearly not real and looks like something you might expect from compensation issues from fluorescence flow.

I've started to think that it might be due to running cells at too high a concentration but I'm not sure. I aim for running 500,000/mL on the low flow rate recommendation and gating on these cells just shows that they are positive for everything they should be positive for and aren't always positive for everything. Does anyone have an idea what this might be?

Screen Shot 2015-03-23 at 5.48.18 PM.png
Screen Shot 2015-03-23 at 5.48.18 PM.png (65.08 KiB) Viewed 8643 times
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komalkumaralienboy

Contributor

Posts: 44

Joined: Tue May 20, 2014 2:24 pm

Location: Linkoping University, Sweden and Finnish Institute of Molecular Medicine, Finalnd

Post Tue Mar 24, 2015 8:35 am

Re: Odd double positive staining

Hi Nick,

Go through my post and responses: [Does anyone experienced spill over or contamination like this]
U will get some ideas what it would be.
But I believe this problem might closely related to the machine and the staining protocol
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ndawson

Participant

Posts: 13

Joined: Wed Jan 08, 2014 4:40 pm

Post Tue Mar 24, 2015 1:30 pm

Re: Odd double positive staining

Thank you for your comment Komal. I read through the other post and the similar staining in that case seems to center around a bead purified population. However, these cells are not bead-purified, they are just cryopreserved PBMC that were isolated with Ficoll. What's more perplexing is that when I do the staining multiple times on the same donor, about 1 in every 3 runs will have this odd staining.

To be clear on my process:
- Isolate PBMC
- aliquot and freeze in LN2
- thaw on staining day
- stain surface
- fix
- perm
- stain IC
- fix/perm- intercalator O/N
- run on CyTOF2
- analyze on FlowJo

Any further insights?
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komalkumaralienboy

Contributor

Posts: 44

Joined: Tue May 20, 2014 2:24 pm

Location: Linkoping University, Sweden and Finnish Institute of Molecular Medicine, Finalnd

Post Tue Mar 24, 2015 2:14 pm

Re: Odd double positive staining

Hi Nick,
are u using FC block.

What about Fixation and Perm buffer, are u using PFA, and Methanol. How do u fix and perm.
Titrate the Abs once .
I would suggest u to split the sample tubes, one goes to CyTOF and one goes to flow cytometry same day .

Gating strategy is important, keep away the doublets.

Just now Andrius Serva from fluidigm called me and gave some tips about dilution of the samples before running CyTOF. may be u could dilute the cells at different concentrations , (Eg: 0.1 million/ml, 0.2 milliom/ml) and run them in CyTOF
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue Mar 24, 2015 3:36 pm

Re: Odd double positive staining

Hi Nick,

Please show us plots of your gating strategy; that can aid us in helping troubleshoot with you.

Also, information about your panel would be helpful, to look for any potential spillovers.


When you're running these samples, what does your background look like? In other words, do you have streaking in any channel(s)?


-Mike
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Ofir

Master

Posts: 75

Joined: Thu Nov 07, 2013 12:46 pm

Location: US, CA

Post Tue Mar 24, 2015 6:03 pm

Re: Odd double positive staining

Hi Nick,
You can upload an FCS file if you would like. That way we can help more directly with investigating the issue.
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mkunicki

Participant

Posts: 17

Joined: Wed Apr 15, 2015 8:43 pm

Post Fri May 06, 2016 6:57 pm

Re: Odd double positive staining

Hi Nick,

Funny bumping into you on Cytoforum!

I was wondering if you ever came to a conclusion on that CD127hi double positive population?

I have seen the same sort of staining (as shown here), but I see it with only one of my frozen healthy donor samples. The donor had other irregularities such as low CD4 count, and high %CD161+ in CD8 T cells which left me to believe this donor may have been ill at the time of blood draw. Considering isotopic impurity, doublet-discrimination, recurrence between experiments, and running multiple healthy donors in parallel, I believe this CD127+++ population had something to do specifically with this donor or sample prep.

I am curious if you'd be willing to discuss your sample processing & freeze/thaw protocol? It would be interesting for our group too to pin-point this issue. For example, one thing we see is perturbations in CXR or CD127 signal with longer/stronger fixation steps, and freezing has had a similar affect (al beit to a lesser extent).


Hope this helps,
Attachments
Screen Shot 2016-05-06 at 11.29.15 AM.png
CD127+++ events
Screen Shot 2016-05-06 at 11.29.15 AM.png (40.05 KiB) Viewed 8201 times
Screen Shot 2016-05-06 at 11.35.34 AM.png
Doublet-discrimination
Screen Shot 2016-05-06 at 11.35.34 AM.png (17.09 KiB) Viewed 8201 times
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AdeebR

Grand master

Posts: 169

Joined: Thu Mar 13, 2014 5:58 pm

Location: NYC

Post Fri May 06, 2016 9:11 pm

Re: Odd double positive staining

Hi folks,

Are you filtering your antibody cocktails prior to staining? This has typically resolved a lot of these weird double positive staining issues for us.
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC

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