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Viability Staining on tissue samples

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Oshbosh

Participant

Posts: 4

Joined: Wed Feb 24, 2016 1:56 pm

Post Thu Feb 25, 2016 10:34 am

Viability Staining on tissue samples

Hi team,
I've been having trouble with Live/Dead discrimination on samples derived from the uterus. So far we have been using Cisplatin which works well on PBMCs but not on cells isolated from the tissue. We are finding that we have a very high background on nearly all the cells, however some are more or less cisplatin "bright". We don't think the cells are all dead as once you gate onto potential live cells, the other Antibody stains look tidy. What's more Flow experiments done in parallel do not share the same problem, there is good viability. This problem arises when cells are stained fresh and also after freezing.

1. I was wondering if anyone else had experienced similar issues?

2. What viability stains do other tissue users find work?

Cheers
Osh
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Feb 25, 2016 4:59 pm

Re: Viability Staining on tissue samples

Hi Osh,

Could you give us more detail?

1. What does your staining (both cisplatin and antibody) look like? Ideally, show us Total cells (Live+Dead), Live, and Dead. For antibody, it would also be helpful to see the same cells in plots like CD3+CD20+ that *aren't* biologically meaningful and which are usually more prevalent in Dead cells.
2. Similar plots for your flow data.
3. What is your uterine tissue dissociation protocol? Have you put the PBMCs through the same protocol to confirm that you aren't killing the cells?

Regarding alternative live-deads: the HIMC usually still uses the metal-loaded maleimide-DOTA from the early days of CyTOF, such as Newell et al Immunity 2012.

For more info, you can check out the end of: http://www.bio-protocol.org/e1382


Mike
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irinafer

Participant

Posts: 1

Joined: Sun Feb 21, 2016 8:35 pm

Post Thu Feb 25, 2016 6:03 pm

Re: Viability Staining on tissue samples

Hi Osh,

We are just starting to use CyTOF to look at immune infiltration in solid tumors, so we have been staining many different tissues like:breast, melanoma,lung...
We use cysplatin and I did have notice that I get two different populations of Cysplatin positive after gating on my singlets. There is always a very bright Cysplatin and a mid cysplatin. So far, I have been considering the very bright as my dead cells.. We are considering the use of Rhodamine instead of Cysplatin to exclude dead cells.
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CLorang

Participant

Posts: 1

Joined: Thu Feb 05, 2015 8:54 pm

Post Thu Feb 25, 2016 10:40 pm

Re: Viability Staining on tissue samples

Hi,

I am a researcher and technical support resource at Fluidigm and have done quite a bit of cisplatin staining with epithelial cells at Stanford.

How long are your cisplatin duration stains? This could be a very critical step. PBMCs can be stained with cisplatin for up to 5min; however epithelial cells can show some cellular death after a more extended stain such as for 1min-5min incubation could be very critical to your sample.

Also: What Do you mean by "Bright" and "High Background"? - I would expect that you would want the cisplatin signal to be bright for detection of dead cells, what is the background and how high is your signal? Is there bleeding over into neighboring channels?

Did you do a viability check before running? This usually helps with live/dead stain analysis and comparison.

Could you send me an email at Cynthia.lorang@fluidigm.com with more information and possibly some data so I could take a look of what the issue is?

Thanks for the Insight!

Cynthia Lorang
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elinastar

Participant

Posts: 19

Joined: Sun Nov 17, 2013 1:46 pm

Post Fri Feb 26, 2016 6:59 am

Re: Viability Staining on tissue samples

Hi all,
We use Rho/Ir for live-dead. We clearly see a difference in Rho permeability to live cells (pre-fix) between different cell types. Cells isolated from mucosa, cancerous tissues, or post- tissue dissociation protocols are highly Rho-permeable even though we are sure they are viable. In such cases we only gate on Ir.
Elina
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Oshbosh

Participant

Posts: 4

Joined: Wed Feb 24, 2016 1:56 pm

Post Fri Feb 26, 2016 10:32 am

Re: Viability Staining on tissue samples

Thanks for the replies team.

Mike:
1. & 2. I've attached an image comparing cisplatin staining using CyTOF and 2 dead cell markers using FACS.
I'm not sure quite what you meant be CD3+CD20+ plots, could you perhaps rephrase what you had in mind?
3. The uterine tissue dissociation protocol essentially consists of mincing the tissue with scalpels, followed by collagenase digestions followed by lymphoprep separations. I should probably clarify that we are looking at immune cell populations in the uterus rather than stromal etc. We don't think that the protocol is killing the cells as the FACS data suggests decent viability. What's more, cells that have been harvested from the tissue in this way and frozen, can be thawed and work in functional assays.
4. I'm interested in looking into the maleimide-DOTA, do you know if it's possible to buy it metal loaded or does that have been done in house? Also do you know if tissue users have had any success with this?

Irinafer:
Sounds like a similar story to us. What sort of marker are you using in combination with cisplatin to gate out your live/dead? Also how confident are you that this cisplatin dim population are the live cells?
I've just ordered some Rho for live dead discrimination, I'll let you know how I get on after testing it. Although given Elina's comment, it may not be looking soo great.

Elina:
Thanks for sharing. May I ask what tissues you're working on and whether your dissociation protocol is similar to ours (see my response to Mike above). If you look at the attachment I've added, is this a similar sort of staining that you see with Rho?
I'm interested to know how you gate on Ir alone, is that just the same as one would always do?
Also have you had any success with cisplatin or maleimide-DOTA on your Rho-permeable samples.

Sorry for the 20 questions everyone. Look forward to hearing your thoughts.
Cheers
Attachments
Viability staining FACS vs CyTOF.pdf
(212.02 KiB) Downloaded 466 times
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Feb 26, 2016 4:38 pm

Re: Viability Staining on tissue samples

Hi Osh,

Could you show your gating pathway starting from Ungated (raw) cells? For your PBMCs as well?

I'm trying to eliminate any other issues in your Cell ID staining (Ir staining, Event length, etc). I have attached an example, from Ungated through Live Intact Singlets. In parallel, I included CD3+CD20+ gate examples for the same populations.

I asked about CD3+CD20+ plots as a QC example: if you haven't gated out the majority of your debris, doublets, and dead cells, you will have a lot more cell events that aren't biologically possible (eg, something that expresses both CD3 and CD20). It's one quick way to look at the overall efficiency of your top gates: If you see more than a few CD3+CD20+, or CD14+CD20+, or CD3+CD14+ cells, then you immediately know something isn't right about your gating, staining, or sample acquisition. Especially since you said that the cisplatin seems to be working well for PBMCs. Ideally, if you compare the CD3+CD20+ frequency of your PBMCs to your uterine samples as you go through your top gates, you should get around the same percentage of CD3+CD20+ after you gate out all the debris/doublets/dead in both tissues. By adjusting the Live gate to include the Cisplatin-dim population, it should allow you to figure out whether it is enriched in CD3+CD20+ and *should* be discarded, or whether it has no effect on CD3+CD20+ and therefore is probably Live.

In fact, if there's a question about Live-dead staining, it's a cheaper experiment to just use CD3, CD20, CD14, Live-dead, and Ir, and make sure that your "impossible" populations like CD3+CD20+ are under control before going back to your full panel.


Mike
Attachments
Mike-top gates.pdf
(236.5 KiB) Downloaded 444 times
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jcvillasboas

Contributor

Posts: 41

Joined: Fri Apr 03, 2015 3:22 pm

Location: Rochester - MN

Post Fri Mar 04, 2016 5:49 am

Re: Viability Staining on tissue samples

I got quite interested in the discussion as I am going through the same dilemma right now. I am working on lymph node tissue and using cisplatin for viability. At occasions it gives me two clearly distinct populations (cis-dim and cis-high) but sometimes the distinction is not as clear as I wanted. Questions for the group: is there a benefit of live-dead over cisplatin for viability analysis? Is there a reason to include both? Thanks everyone. Best. -JC
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Mar 04, 2016 4:47 pm

Re: Viability Staining on tissue samples

Hi JC,

Could you clarify what you mean by "live-dead over cisplatin for viability analysis"? Cisplatin is usually *used* for live-dead purposes. Did you mean using the Rh103 intercalator, or the maleimide-DOTA?


Osh: sorry, I think I forgot to answer your question about the maleimide-DOTA live-dead. We make our own in-house (see the bioprotocols link from an earlier post), as this allows us to save all the bright Ln channels for antibody markers. Therefore, we buy the unloaded maleimide-DOTA from Macrocyclics, and load it ourselves.

However, Macrocyclics does sell maleimide-DOTA pre-loaded with Tm (Tm-Maleimido-DOTA(X-275) ) or Ho (Ho-Maleimido-DOTA(X-279)). However, those would sacrifice two very bright channels that are probably best left for antibody staining.


One side note: Macrocyclics does also sell p-SCN-Bn-DOTA pre-loaded with Tm, Ho, or Gd. This is similar to the p-SCN-Bn-EDTA that Zunder et al and others have used for Pd-based barcoding. Again, you would be using Tm and Ho channels for barcoding, which seems overkill to me, but doable. *However*, I want to point out again that the Gd construct should *not* be used in CyTOF experiments: the last I heard, it was made with natural-abundance Gd, *not* isotopically-enriched Gd like what Fluidigm (or Trace, or Cambridge Isotopes) sell. This would wipe out up to 7 channels in the 152-160 range.


Mike
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jcvillasboas

Contributor

Posts: 41

Joined: Fri Apr 03, 2015 3:22 pm

Location: Rochester - MN

Post Fri Mar 04, 2016 6:59 pm

Re: Viability Staining on tissue samples

Mike,

Thanks for your message. Let me clarify: when I mentioned live-dead I was referring to the Rh103 intercalator. Are there advantages of one over the other? Thanks a lot. Best.

-JC
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