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cisplatin 198 labelling

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nkhanbham

Master

Posts: 53

Joined: Wed Feb 25, 2015 3:03 pm

Post Fri Sep 14, 2018 10:39 am

cisplatin 198 labelling

I have seen that Fluidigm sell cisplatin 198 isotope (cat 201198). Does anyone know how pure this is? I would be interested to know peoples experience of using it to conjugate antibodies.
Regards,
NK
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Jahangir

Contributor

Posts: 36

Joined: Sun Oct 29, 2017 6:34 pm

Post Fri Sep 14, 2018 11:34 am

Re: cisplatin 198 labelling

Hi NK,

I've actually asked this question to my rep/Technical Specialist. They've said that the Monoisotopic Cisplatins Fluidigm sell commercially have to have a "purity" (I'm assuming here they mean an isotopic purity vs a chemical purity) of 98% or above.

I have conjugated antibodies using the method detailed in the Mei et al. paper (-> https://www.ncbi.nlm.nih.gov/pubmed/26355391) using the 194-Pt but not the 198-Pt. The reason being, I am using the 198-Pt as a live/dead cell marker.

From my experience, due to the extremely intense signal we get in the 193-Ir channel, it bleeds over into the 194-Pt, so whatever antibody you put in there has to compete with that signal from the 193-Ir. Furthermore, because (and as detailed in the paper) this conjugation method uses the free lysines (i think) on the antibody itself to conjugate the cisplatin to vs the use of polymers in the MaxPar Fluidigm protocol, it gives a much weaker signal. So you have to put a really strong and abundant marker (for your experiment/cells/etc) if you do want to conjugate using the 198-Pt.

All of that being said, I haven't actually tried using the 198-Pt - maybe others have tried and they can send their input here.

Hope all of the above helps!

Jahangir
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Khantain

Participant

Posts: 1

Joined: Tue Apr 18, 2017 11:34 am

Location: CEA Fotenay-aux-Roses

Post Fri Sep 14, 2018 12:41 pm

Re: cisplatin 198 labelling

Hi,

I recently used 198Pt from Fluidigm with the protocol from Mei et al. and I got good results with an anti-CD66 antibody (I coupled 100ug of the TET2 clone). I tested this conjugation with the "compensation protocol" from Chevrier et al., 2018 (I recorded beads one by one, not pooled) and I found this 198Pt solution has relatively good purity but contains some 194, 195 and 196 traces. I join a screenshot of the "matrix compensation" generated by CATALYST. The spillover of 198 into the 194 channel is under 1.5% in my panel (CD66 into CD20).

If you are interested I can email you more plots of the stainings with 194 and 198Pt.

Image

Regards,

Quentin
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hartmannfj

Participant

Posts: 7

Joined: Fri Oct 31, 2014 3:51 pm

Post Sun Sep 16, 2018 8:01 pm

Re: cisplatin 198 labelling

Hi there,

I just wanted to mention that when we were using all (194/195/196/198) platin isotopes for live cell barcoding (https://www.nature.com/articles/s41598-018-28791-2), we swapped out the iridium intercalator with the rhodium intercalator (also from Fluidigm). It has a very good signal in 103, and it actually does not bleed much into the nearby channels so it is also compatible with using the palladium channels for something. The protocol is excatly the same, although I used slightly different concentration.

If you want to use all the above mentioned platinum isotopes for antibodies, you could also use a palladium-based live dead reagent (see the above paper) which is quiet cheap and easy to use.

Just thought this might be interesting for anyone trying to use platinum channels for antibodies.

Best, Felix

TLDR; DNA 103Rh, L/D natPd
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nkhanbham

Master

Posts: 53

Joined: Wed Feb 25, 2015 3:03 pm

Post Mon Sep 17, 2018 2:34 pm

Re: cisplatin 198 labelling

Thanks to all for the feedback. I am going to try and conjugate it to CD45. I was a little concerned about how stable the labelling was. Any 198Pt that fell off the CD45 and then binding to proteins non-specifically is obviously not desirable.
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hartmannfj

Participant

Posts: 7

Joined: Fri Oct 31, 2014 3:51 pm

Post Mon Sep 17, 2018 6:37 pm

Re: cisplatin 198 labelling

We have tested specificity and it looked good, no (not more than with X8 polymers) unspecific staining. It should be a covalent bond so I don't think it will come off. Good luck with it!

Felix
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henkmei

Participant

Posts: 16

Joined: Wed Jul 30, 2014 9:44 pm

Post Tue Sep 18, 2018 8:27 am

Re: cisplatin 198 labelling

Hi,

we regularly use platinum labeled Abs in various studies and we still do the labeling as published without any issues.
https://onlinelibrary.wiley.com/doi/ful ... to.a.22778.

Since there is no amplification by polymers as implemented in the MAXPAR strategy, Pt labeled Abs usually show somewhat lower signal intensities.We use them for mainly (human) cell-surface lineage marker Abs CD3,CD8, CD134, CD45, IgD, CD5, CD38, but we also published validation with Abs against cytoplasmic and nuclear targets. We often include them in CD45 or B2M-based cell surface barcoding schemes. All Pt isotopes work similarly well, yet we always think twice when using Pt194 because of potential spillover from Ir. This was mainly an issue in our CyTOF v1 times, but did at least not terribly resurface with the Helios. We also try to limit Ir staining intensity in respective assays.

Since Pt is an integral part of cisplatin and most cisplatin attaches to the Ab via free thiols (omitting reduction w TCEP strongly interferes with Ab labeling success), the vast majority of Pt should be covalently bound to the Ab, so I would not expect issues with cross contamination. We found that unlike other conjugates, platinum conjugates show practically no or only very little signal loss over a measurement time of several hours.

Good luck and best wishes

Henrik
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XMUCyTOF

Participant

Posts: 7

Joined: Tue Aug 07, 2018 3:09 am

Post Thu Jul 04, 2019 6:20 am

Re: cisplatin 198 labelling

Hi,

What's the concentration of the 198Pt from Fluidigm ? I'd like to use 194Pt ,195Pt,196Pt and 198Pt at the same time, will it work?


Khantain wrote:Hi,

I recently used 198Pt from Fluidigm with the protocol from Mei et al. and I got good results with an anti-CD66 antibody (I coupled 100ug of the TET2 clone). I tested this conjugation with the "compensation protocol" from Chevrier et al., 2018 (I recorded beads one by one, not pooled) and I found this 198Pt solution has relatively good purity but contains some 194, 195 and 196 traces. I join a screenshot of the "matrix compensation" generated by CATALYST. The spillover of 198 into the 194 channel is under 1.5% in my panel (CD66 into CD20).

If you are interested I can email you more plots of the stainings with 194 and 198Pt.

Image

Regards,

Quentin
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mleipold

Guru

Posts: 2086

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Jul 08, 2019 3:28 pm

Re: cisplatin 198 labelling

Hi,

If you look at the TDS for the 198Pt cisplatin, it says on p. 1 that the master stock is 1 mM:

"(1000X dilution of 1 mM stock solution, i.e.,1 μL Cell-ID Cisplatin-198Pt added to 1 mL of cell suspension)."


Mike

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