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Discriminate cells treated with cisplatin

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It is fine to promote your company's reagents. Just make sure they are relevant to CyTOF, and do so in moderation and style :-)
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slamorte

Participant

Posts: 3

Joined: Mon Oct 24, 2016 2:30 pm

Post Mon Oct 24, 2016 9:19 pm

Discriminate cells treated with cisplatin

Hi everyone! I'm using Cisplatin (P4394 from Sigma) to treat my cells. I'd like to take advantage of this to discriminate between treated and non treated cells. How do I know if my Cisplatin is mono-isotope? In case is a mix of isotopes, how can I know its composition ? I know that the drug I'm using is 65% 195Pt, but I would like to know if there is any 194pt and in which percentage. So that I can use the other isotope to discriminate live and death cells. thanks everyone.
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AdeebR

Grand master

Posts: 169

Joined: Thu Mar 13, 2014 5:58 pm

Location: NYC

Post Wed Oct 26, 2016 5:11 pm

Re: Discriminate cells treated with cisplatin

Chances are good that commercial cisplatin you buy from a non-Fluidigm source is not going to be monoisotopic. I would expect the isotopic distribution to mirror that of natural abundance platinum:
192Pt ~<1%
194Pt ~33%
195Pt ~34%
196Pt ~25%
198Pt ~8%

You can determine this empirically by treating your cells with your drug and measuring the signal intensity in all the above channels.

If you wanted to also use monoisotopic cisplatin for live/dead discrimination I would use Pt198, but I think you'd be much better off using Rh103.

Adeeb
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC
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Chowduck

Contributor

Posts: 29

Joined: Wed Nov 19, 2014 4:39 pm

Post Wed Oct 26, 2016 5:25 pm

Re: Discriminate cells treated with cisplatin

Hi Sara,

Unless the vendor specifically states that the Pt has been isotopically enriched, like Adeeb said I would expect it to be distributed over the naturally found abundances of Pt. You could check by running a very dilute cisplatin solution, I suspect something like 1pg/ml or more dilute should be in the ball park. (Someone else might have a better idea of the appropriate dilution). Then look at the TOF graph, or masses per reading graph with the Pt channels selected that Adeeb listed to compare the relative values of Pt isotopes present. The TOF graph should give you some values if you mouse over the peaks.

Regards,
-Greg
UHN/Sickkids FCF
Toronto, Canada
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Chowduck

Contributor

Posts: 29

Joined: Wed Nov 19, 2014 4:39 pm

Post Wed Oct 26, 2016 9:51 pm

Re: Discriminate cells treated with cisplatin

Hello again,

I just ran the cisplatin we use (Cedarlane/Biovision cat#1550-1000) in solution mode at 1E-7M (30 ng/mL), attached is the TOF graph. Using the area under the curve I get the following rough estimate of the isotopic abundance:

192Pt 0.6%
194Pt 31.7%
195Pt 34.2%
196Pt 26.1%
198Pt 7.5%

Cisplatin 10e-7M.png


Probably could have saved myself some trouble and used the peak height rather than area and come to the same conclusion.

Regards,
-Greg
UHN/Sickkids FCF
Toronto, Canada
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slamorte

Participant

Posts: 3

Joined: Mon Oct 24, 2016 2:30 pm

Post Thu Oct 27, 2016 2:38 pm

Re: Discriminate cells treated with cisplatin

AdeebR wrote:Chances are good that commercial cisplatin you buy from a non-Fluidigm source is not going to be monoisotopic. I would expect the isotopic distribution to mirror that of natural abundance platinum:
192Pt ~<1%
194Pt ~33%
195Pt ~34%
196Pt ~25%
198Pt ~8%

You can determine this empirically by treating your cells with your drug and measuring the signal intensity in all the above channels.

If you wanted to also use monoisotopic cisplatin for live/dead discrimination I would use Pt198, but I think you'd be much better off using Rh103.

Adeeb



Thanks a lot!!
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slamorte

Participant

Posts: 3

Joined: Mon Oct 24, 2016 2:30 pm

Post Thu Oct 27, 2016 2:39 pm

Re: Discriminate cells treated with cisplatin

Chowduck wrote:Hello again,

I just ran the cisplatin we use (Cedarlane/Biovision cat#1550-1000) in solution mode at 1E-7M (30 ng/mL), attached is the TOF graph. Using the area under the curve I get the following rough estimate of the isotopic abundance:

192Pt 0.6%
194Pt 31.7%
195Pt 34.2%
196Pt 26.1%
198Pt 7.5%

Cisplatin 10e-7M.png


Probably could have saved myself some trouble and used the peak height rather than area and come to the same conclusion.


Hi Greg,

I've submit my sample to your facility, I'm curious to compare the results with yours. Thank you very much!!
Regards,
-Greg
UHN/Sickkids FCF
Toronto, Canada
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dtdavani

Participant

Posts: 12

Joined: Fri Sep 11, 2015 9:52 pm

Post Fri Oct 28, 2016 4:39 pm

Re: Discriminate cells treated with cisplatin

Hi,

Testing drug treated cells shows high MMI in
190pt 10
192Pt 800
194Pt >10000
195Pt >10000
196Pt >10000
198Pt >10000

This data suggests that 198 is not an option. Better choice would be monoisotopic 190pt dead/live or ,as Adeed said, alternatives such as Rh103.
I would try the same treatment and staining including Rh103.

Best of Luck
Dariush
UHN/Sickkids FCF
Toronto, Canada
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GregBehbehani

Master

Posts: 85

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Mon Oct 31, 2016 3:48 pm

Re: Discriminate cells treated with cisplatin

I would agree with everyone else, you could use Pt190 (though I'm not sure where you would get it from). Alternatively, you can use the original barcoding protocol (Bodenmiller et al, 2012, Nature Biotechnology) to turn any metal channel into a live-dead stain (essentially any barcoding reagent can be used as a live-dead stain).

If Rh103 is working in your hands that would certainly be the easiest, but I would make sure that it doesn't wash off the cells as it's interaction isn't supposed to be covalent. As long as it's consistent in the staining protocol you're using, it should be fine.

Good luck,

Greg

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