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Rhodium 103Rh antibody labelling

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MCOlivier

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Posts: 31

Joined: Mon Oct 05, 2015 9:48 am

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

Post Fri Feb 23, 2018 10:32 am

Rhodium 103Rh antibody labelling

Hi all,

Has someone ever tried loading 103Rh on maxpar polymers for antibody staining ? I looked around in this part of the forum, but didn't notice such a topic.

Thanks +++ in advance (for all previous and future comments ^^)

Olivier
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mleipold

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Location: Stanford HIMC, CA, USA

Post Fri Feb 23, 2018 4:12 pm

Re: Rhodium 103Rh antibody labelling

Hi Olivier,

I haven't tried loading MAXPAR with Rh103. A couple years ago, Henrik and I tried loading Rh103 into the maleimide-DOTA (I use it as a live-dead, though Bodenmiller et al used it as a BC reagent). We used Sigma #450286, as the high-purity chloride salt, and dissolved into L-buffer.

At best, we got mixed results. We compared it to our regular In115-mDOTA, and in some cases, it compared well, but in other preps, it didn't (had no signal on the same cells that In115 was showing signal).

Granted, DTPA (on the MAXPAR) and DOTA are different chelators, but they usually bind similarly. If you do want to try it, I'd suggest a lot of validations (including stability testing; just because you get it in there doesn't always mean it will stay in under staining conditions).


I think Henrik tried again after he went back to Germany; maybe he'll comment further?


Mike
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MCOlivier

Contributor

Posts: 31

Joined: Mon Oct 05, 2015 9:48 am

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

Post Mon Feb 26, 2018 8:24 am

Re: Rhodium 103Rh antibody labelling

Hi Mike,

Thanks a lot for this help ! I will wait for Henrik comments and see.
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jlederer

Participant

Posts: 8

Joined: Thu Feb 08, 2018 2:42 pm

Post Mon Feb 26, 2018 4:13 pm

Re: Rhodium 103Rh antibody labelling

Hi Olivier and Mike,

We have been working on labeling Abs with Rhodium as well. I think it is worth the effort. We did get it to load into the MaxPar reagent, but it was a low signal. We will be optimizing and retesting. I will share with the Forum whatever progress we make with this.

Best regards,

Jim
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MCOlivier

Contributor

Posts: 31

Joined: Mon Oct 05, 2015 9:48 am

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

Post Tue Feb 27, 2018 10:34 am

Re: Rhodium 103Rh antibody labelling

Hi Jim,

Thx for sharing your experience !
I will see how I can manage the panel I'm designing, but I think I will try to conjugate 103Rh too ^^

I'll let you now if any progress/outcome.
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MCOlivier

Contributor

Posts: 31

Joined: Mon Oct 05, 2015 9:48 am

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

Post Mon Jul 15, 2019 2:28 pm

Re: Rhodium 103Rh antibody labelling

Hi all,

I tested 103Rh coupling this day. Here are the resultas I get on anti-Human CD8 (Biolegend 344727) which are pretty comparable, but intensity is week.
I also put a 195Pt labeled CD8 (same clone).

I basically followed the Nolan's Lab protocol for custom AB labelling regarding 103Rh coupling.

What are your advice for increasing resolution ? Increasing metal concentration during polymer loading (5µL 50mM 103Rh laoded actually) ?

Best regards !

Olivier
Attachments
103Rh coupling vs 195Pt.pdf
(221.67 KiB) Downloaded 90 times
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Jahangir

Contributor

Posts: 36

Joined: Sun Oct 29, 2017 6:34 pm

Post Tue Jul 16, 2019 11:13 am

Re: Rhodium 103Rh antibody labelling

Hi Olivier,

Thanks for sharing your data/experience with us.

I hope this isn't an ignorant reply to your post, and I'm assuming you've already tried this, but can you not just increase the volume of CD8-103Rh in your cocktail? Would there be any harm in doing that?

Many thanks,

Jahangir
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MCOlivier

Contributor

Posts: 31

Joined: Mon Oct 05, 2015 9:48 am

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

Post Tue Jul 16, 2019 11:22 am

Re: Rhodium 103Rh antibody labelling

Hi Jahangir

Thank's for reading :)

That's what I'll try next week, but I already put it at 1µg anti-CD8/tube (3.10^6 cells max in 100µL, classical indeed) and I think we already saturate the CD8s, as usually, I work with 0.2µL in flow Cytometry...

Would it be worthy trying to increase loading time on polymer ? Not sure of it.
And what about doubling the quantity of polymer/antibody ? Not sur either, as we are supposed to saturate the AB with the polymer prodived in those small Fluidigm tubes...
To be continued then ;-)

Best regards

Olivier

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