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Suppression or another issue on CyTOF1?

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salong

Participant

Posts: 6

Joined: Thu Jun 11, 2015 2:47 pm

Post Tue Sep 29, 2015 5:51 pm

Suppression or another issue on CyTOF1?

We would like to understand whether loss of staining is due to suppression on our CyTOF1 or something else.

We have CXCR3 in 156. When stained as part of a MMM panel, we detect it fine but loose staining in the panel with all Ab's. We have tried pre-staining with CXCR3 in the all panel testing whether steric hindrance contributes - this didn't help. We also have other panels with CXCR3 in 156 and get similar results, so likely not a +/-1 contaminant. We see this with some other Ab's primarily in the mid-range "sweet-spot" which is why we have been wondering about suppression which occurs more prominently in this mass range but are concerned that dropping nearby string stainers does not fix the problem.

We tested our panel on a CyTOF1.5 and got much better staining, although not completely comparable to MMM. Leaving me with 2 main questions:

1. What is the source of our loss of CXCR3 staining on our CyTOF1? Suppression? Poor detector? other machine issues? other panel issues?
2. Does CyTOF 1.5 improve resolution that well or are there other things we also need to address to improve performance of our CyTOF1?

Even if you don't have answers to these questions, it would be great to hear if others have seen similar issues.

Thanks,
Alice

Is it suppression.pptx
representative data assessing loss of CXCR3 156 staining in full panel, but not MMM
(231.97 KiB) Downloaded 316 times
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue Sep 29, 2015 6:31 pm

Re: Suppression or another issue on CyTOF1?

Hi Alice,

1. What software versions are you running on each of your machines?

2. For your v1 vs v1.5 comparison, was this the same sample stained, then *split* and run on each machine on the same day? Or are these representative but separate staining experiments?
-Part of why I ask is the 50% reduction difference between machines in your CD127+ populations in the CD8 vs CD127 plots on page 1.

3. Someone more familiar with signal suppression (Nolan Lab? Fluidigm?) should chime in on this, but my understanding is that signal suppression is primarily in +/- 1 mass. If so, Mass M would partially suppress M-1 and M+1, but that this only becomes significant when the signal at M is particularly bright.
-Please post your entire panel used in these experiments, so we can see what markers you have neighboring these trouble spots.
-Are the plots you're showing us of total Live Intact Singlets (ie, debris, doublets, dead cells, etc removed)? Could you show us what your gating pathway is down to the population you're plotting?


Mike
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salong

Participant

Posts: 6

Joined: Thu Jun 11, 2015 2:47 pm

Post Thu Oct 01, 2015 7:24 pm

Re: Suppression or another issue on CyTOF1?

Hi Mike,

Thanks for your reply.

1. I'm not sure if I understand your question about software. We're using software from Fluidigm for the CyTOF1 and CyTOF1.5 platforms for acquisition. Do they differ?
2. Yes, the same sample was stained and then split and run on different machines the same day.
3. Fluidigm is helping with the suppression question, but I am curious to know whether other users are also seeing similar staining patterns/issues and if so, do they know the cause and/or a solution?

The entire panels are below:
MMM1 (A) includes Abs: CD49D 141, CCR6 142, LAIR1 145, CD16 148, CD123 151, CD45 154, CRTH2 160, CD3 163, CD24 166, CD25 169, IgM 172, and CD4 175.
MMM2 (B) includes Abs: CD33 143, IgD 146, CCR4 149, CD45RO 152, CD27 155, CD10 158, CD15 164, CD38 167, CD122 170, CCR3 173, CD56 176.
MMM3 (C) includes Abs: CD62L 144, CD20 147, CCR7 150, CD45RA 153, CXCR3 156, CD161 159, CD11c 162, CD127 165, CD8 168, CXCR5 171, HLADR 174.
All has everything together.

Plots are of total live intact singlets. I will post gating pathway as soon as I've pulled it together in a file easy to share.

Thanks,
Alice
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Oct 05, 2015 4:04 pm

Re: Suppression or another issue on CyTOF1?

Hi Alice,

You can check the software version. If I remember correctly in those versions of the software, you can see which version is running on the computer by opening the login screen (like you were going to switch to service mode). It should be listed there.


Mike

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