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Getting monoisotopic cisplatin Pt195 and 196

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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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bsahaf

Participant

Posts: 1

Joined: Thu Sep 13, 2018 10:19 pm

Post Wed Sep 19, 2018 9:09 pm

Re: Getting monoisotopic cisplatin Pt195 and 196

Hi Muharram,
I hope it is not too late, please Count our lab ( Correlative Science unit) in,
regards,
Bita
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MCOlivier

Contributor

Posts: 32

Joined: Mon Oct 05, 2015 9:48 am

Location: European Genomic Institute for Diabetes, Universitity of Lille, Institut Pasteur de Lille, France

Post Mon Sep 24, 2018 6:40 am

Re: Getting monoisotopic cisplatin Pt195 and 196

Hi Muharrem,

Do you have any idea how long it will take to get those two isotopes, and who you will proceed for facturation/shipment ?

Very impatient I am :-p
Kind regards

Olivier
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Lauren

Participant

Posts: 1

Joined: Fri Nov 02, 2018 7:56 am

Post Fri Nov 02, 2018 10:36 pm

Re: Getting monoisotopic cisplatin Pt195 and 196

Hi Muharrem,

I know we're really late to the game, but our group (the Spitzer Lab) recently set up shop at UCSF and would be extremely interested in joining in as well. Is it too late at this point? Thanks in advance for considering!

Best regards,
Lauren
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JamesW

Contributor

Posts: 26

Joined: Tue Nov 21, 2017 6:59 am

Post Tue Nov 20, 2018 3:44 am

Re: Getting monoisotopic cisplatin Pt195 and 196

I may have missed the boat on this one but I did want to ask if you guys have concluded this successfully and who the vendor is, as I am quite interested in obtaining 195, 196 Cisplatin as well.

Best,
James
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MCOlivier

Contributor

Posts: 32

Joined: Mon Oct 05, 2015 9:48 am

Location: European Genomic Institute for Diabetes, Universitity of Lille, Institut Pasteur de Lille, France

Post Thu Jul 25, 2019 11:09 am

Re: Getting monoisotopic cisplatin Pt195 and 196

Hi Forum ^^

Just to give a little feedback, those 195-Pt and 196-Pt are functionnal !

Thanks to all of those who made this acquisition possible.
Olivier
Attachments
Test 195-196-Pt 15-07-2019.pdf
(251.62 KiB) Downloaded 157 times
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JamesW

Contributor

Posts: 26

Joined: Tue Nov 21, 2017 6:59 am

Post Fri Aug 02, 2019 8:53 am

Re: Getting monoisotopic cisplatin Pt195 and 196

Dear Olivier,

Thanks for letting us know. We have also just used the 195 and 196Pt to make CD45 antibodies and at first glance of the staining they seem to have worked pretty well. Its a relief as actually we had some trouble with the shipment getting stuck at customs so when the Cisplatins finally arrived they had been warm for some time.

Thanks to everyone involved and Muharrem in particular.

Best,
James
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dahern

Participant

Posts: 12

Joined: Tue Mar 31, 2015 3:45 pm

Post Fri Aug 02, 2019 9:27 am

Re: Getting monoisotopic cisplatin Pt195 and 196

Just wondering how are people storing their new Cisplatins? What temperature and have you split the stock into smaller aliquots?

Thanks!

David.
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Jahangir

Contributor

Posts: 37

Joined: Sun Oct 29, 2017 6:34 pm

Post Fri Aug 02, 2019 11:52 am

Re: Getting monoisotopic cisplatin Pt195 and 196

Hi Guys,

Olivier: Thanks for sharing your data! It actually looks really good. Did you label the antibodies according to the protocol detailed in the Mei et al, Cytometry A, 2015 paper?

David: I received the Cisplatins a few weeks ago and I made a 1mM stock from the 10mM stock they provided us. I then aliquoted the 1mM into stock in 10uL aliquots and stored all of those in the -20c (essentially mimicking what Fluidigm sell us and advise us to do with the commercially available Cisplatins (the monoisotopic -194 and -198) we buy from them). With the rest of the 10mM stock, I then placed that into the -80c freezer for long term storage. To my (limited) knowledge and experience, these small molecules can last for quite a while in the -20c freezer, so they should last longer in the -80c. Let me know what you think!

Best regards,

Jahangir
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kanthsmiles

Participant

Posts: 7

Joined: Sun May 11, 2014 2:41 pm

Post Thu Aug 08, 2019 2:00 pm

Re: Getting monoisotopic cisplatin Pt195 and 196

Hi guys

I have a question regarding the conjugation of Cisplatins (that many of us received a few weeks ago from Petr) to Antibodies. I guess the protocol followed for coupling is the one detailed in Mei et al, Cytometry A, 2015 paper..

I also saw that in Hartmann et al, Scientific Rep, 2018 paper, they say that 1 mM stock solutions of isotopically enriched cisplatin in DMSO was pre-conditioned for 48 h at 37 °C [ref.43] and then added to the reduced Ab. I'm wondering if everyone does this or not? Its not mentioned in the Mei et al paper as I see.

Can someone shed light on the importance of this pre-conditioning step? I understand that its more of a concern when Cisplatin is used as a drug in patients due to toxicity effects but I guess in these conjugations as well, it might be pretty important if DMSO displaces the complex structure..

Regards
Kanth
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mleipold

Guru

Posts: 2095

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Aug 08, 2019 3:22 pm

Re: Getting monoisotopic cisplatin Pt195 and 196

Hi Kanth,

We did not perform the pre-conditioning in the original Mei et al paper. Among other things, we didn't find the ref 43 that Felix cited (https://cancerres.aacrjournals.org/cont ... /3913.long).

Based on what Felix wrote, though, it doesn't seem to be *required*, but I don't think it would be bad for the conjugation. In fact, in the discussion in ref 43, there's this comment: "For example, a crystallographic study of cisplatin and carboplatin binding to histidines of hen egg-white lysozyme found one platinum bound to His15 when cisplatin was dissolved in aqueous medium. When dissolved in DMSO, two platinum atoms bound to His15, leading to the conclusion that DMSO could facilitate platinum-protein binding (49)."


I think some of it comes down to the fact that the *cytotoxicity* of cisplatin and the *chemical reactivity* of cisplatin are technically different things. At least a large part of the *cytotoxicity* is due to the formation of crosslinks, which is dependent on the cis structure and replacement of both Cl ligands by something else (often the exocyclic amines of Guanines, to form G1-NH2-mu(Pt-(NH3)2)-NH2-G2, where mu means it's bridging between the two Guanines, and the two NH3 are remaining ligands from the cisplatin (cis-PtCl2(NH3)2) after the chlorides are displaced.

So, in order to form a *crosslink*, you need both Cl's (technically, inside the cell the Cl's can be replaced by H2O ligands, but you still need both Cl's to start with). Therefore, if you don't have that due to ligand replacement, you can't form a crosslink.

However, to just *label* something with cisplatin, you just need *at least one* cisplatin ligand to be replaced by a biological amine (or to a lesser extent, thiol).....it doesn't matter whether it forms a crosslink (2 ligands) or whether it forms a terminal/pendant adduct (1 ligand). I think the preconditioning in the DMSO helps reduce the cytotoxicity just to make it friendlier to handle in the lab.....


Mike
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