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Cisplatin Staining Problem

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mtl536

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Posts: 2

Joined: Sat Nov 21, 2020 6:58 pm

Post Sat Nov 21, 2020 8:46 pm

Cisplatin Staining Problem

Hello!

I have run into an issue with my sample analysis when trying to gate for live cells. After staining with the cisplatin live/dead from fluidigm, all of my cells end up being 195pt+ as if they are all dead?

I am running pediatric blood samples. My samples are frozen at -80 in PROT-1 stabilizer. I thaw them over ice. I have run a test on a fresh sample which stained without issue. I used the same lysis buffer on both (ACK Lysis buffer). I perform a Trypan blue cell count prior to staining and just prior to running the samples and I have live (or intact) cells at both steps by that stain.

I'm wondering if anyone else has encountered this problem or if anyone might be able to offer a solution or resources that can help me figure out what is going on.

Thanks!
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cguidos

Contributor

Posts: 28

Joined: Tue Nov 18, 2014 3:10 am

Post Sun Nov 22, 2020 9:32 pm

Re: Cisplatin Staining Problem

Hi Matthew. You can't use Cisplatin to stain fixed cells because the fixation permeabilizes them and all cells are thus "dead". In flow we have options for fixable live/dead stains but I am not aware of such reagents for CyTOF. Your only option would be to Cisplatin stain and wash before storing in PROT1 stabilizer but that is seldom feasible

Cynthia
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bbengsch

Participant

Posts: 4

Joined: Thu Jun 19, 2014 12:39 pm

Post Mon Nov 23, 2020 11:26 am

Re: Cisplatin Staining Problem

In our hands the MM-DOTA approach works well to distinguish Live/dead cells. This is the protocol by Leipold & Maecker : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4933512/
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mleipold

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Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Nov 23, 2020 3:28 pm

Re: Cisplatin Staining Problem

Hi Matthew,

To expand on what Cynthia wrote:

PROT1 stabilizer is a fixative. After treating with that, your cells are no longer alive/viable, regardless of what your Trypan is telling you.

Remember, different livedeads act differently. Small molecule live-deads like cisplatin (300Da, mostly from the Pt) can enter a fixed cell: the fixation process "opens up" the membrane enough for them to slip in. Trypan blue on the other hand is large enough (MW 872Da) and rigid; fixation alone often isn't enough for it to enter cells reliably....you usually need a perm, or large membrane disruptions that come with a true dying cell.

In short: it's possible for cisplatin to say one thing and trypan to tell you something different. Regardless, you can't use cisplatin on fixed cells.


Mike
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mtl536

Participant

Posts: 2

Joined: Sat Nov 21, 2020 6:58 pm

Post Mon Nov 23, 2020 6:08 pm

Re: Cisplatin Staining Problem

Thanks very much for your help!

If I'm unable to determine live from dead cells via this method, are there other ways that I can assure that the signals that I'm getting from my sample are actually the cells of interest as opposed to indiscriminatley stained dead cells?

Thanks again, this has been very helpful!
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eganio

Contributor

Posts: 21

Joined: Wed Nov 07, 2018 8:00 pm

Post Mon Nov 23, 2020 6:56 pm

Re: Cisplatin Staining Problem

Hi Matthew,

Our lab uses cleaved PARP (cleaved at D214) to detect dead/dying cells amongst a population of fixed cells, although this analysis is limited to apoptotic cells. Unfortunately, you're rather limited with regard to detecting dead cells once the cells have been fixed (as far as I know).

-Ed
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Nov 23, 2020 6:58 pm

Re: Cisplatin Staining Problem

With your current frozen samples, you might be able to add in an apoptotic marker like cleaved-caspase (https://www.fluidigm.com/reagents/prote ... b--50tests)

If you're still collecting (drawing and freezing) your samples, you might be able to do a quick Rh103 intercalator incubation like in the MDIPA kit protocol, *prior to* adding the PROT1: https://www.fluidigm.com/reagents/prote ... ling-assay

However, while I've used the Rh103 already inside the MDIPA kit for WB which was then fixed with PROT1, I haven't tried just adding the Rh103. You might reach out to your Fluidigm FAS, as they might have some info on concentrations from their MDIPA development work....


Mike

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