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Indium chemistry

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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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nkhanbham

Master

Posts: 53

Joined: Wed Feb 25, 2015 3:03 pm

Post Wed Mar 28, 2018 10:38 am

Re: Indium chemistry

The compensation idea sounds great. Can kappa beads labelled with metals be reused or frozen in aliquots and stored for later use? Would save a huge amount if time if that was possible.
Naeem
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Mar 28, 2018 11:01 pm

Re: Indium chemistry

Hi Jahangir,

Both the Rh103 and Ir intercalators work equally well for DNA. Just realize that the Ir intercalator has *two* isotopes (Ir191 vs Ir193 that many of us use as bivariates), whereas the Rh103 only has 103 (100% nat abund.).

You can definitely buy highly pure RhCl3 (eg, Sigma #450286), and it dissolves easily into L buffer. I haven't tried loading MAXPAR polymer with it, though I think some other people have.

I did try loading it into mDOTA to make a live-dead; it didn't seem to work as well as In115 (lower signal for same concentration, even accounting for the lower sensitivity of 103 vs 115). I didn't try optimizing it (heating while loading, etc). So, if you want to give it a try, let us know how it turns out!


Mike
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nkhanbham

Master

Posts: 53

Joined: Wed Feb 25, 2015 3:03 pm

Post Mon May 14, 2018 1:49 pm

Re: Indium chemistry

I am planning on conjugating CD57 to 115In. However the CD57 clone we have is an IgM (HCD57). I see that Fluidigm also sell the IgM clone conjugated to various metals. Given that we have been told MaxPar conjugation doesn't work with IgM can anyone clarify if it works with this antibody?
Thanks,
Naeem
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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 May 14, 2018 2:07 pm

Re: Indium chemistry

Hi Naeem,

Some people have had trouble with IgM conjugations not working, and there is a suggestion that the antibody pentamer is probably falling apart during the labeling reaction (which make sense, but I'm not sure anyone has proven that). That said, if you're using the same antibody clone as Fluidigm, it should work fine (perhaps the affinity of the antibody is high enough that it functions fine as a monomer).

Just remember how much "dimmer" In115 will be, it will only work well if you see high signal with the Fluidigm antibodies.

best of luck,

Greg
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon May 14, 2018 2:47 pm

Re: Indium chemistry

Hi Naeem,

I make an in-house conjugate using the HCD57 anti-CD57 clone; it works perfectly fine on Indium.

Whether the TCEP is short or mild enough that the IgM doesn't fall apart, or whethere the monomers are high-affinity, I don't know (I haven't tried running it on a native PAGE or size-exclusion). But it does work fine.

In fact, I know some people who use that clone on 89Y: In113 still gives me something around 1000 Median for the pos population on CD8 and NK cells. Especially since CD57 is nicely bimodal, that still gives you a lot of signal to work with.


Mike
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taxkourel

Participant

Posts: 14

Joined: Sun Sep 03, 2017 3:46 pm

Post Mon May 14, 2018 2:52 pm

Re: Indium chemistry

I 've used this clone with the MaxPar conjugation kit on 152Sm and it works great. Lots of signal too (on PBMCs), had to dilute it 8 fold so have plenty of antibody left. 6 months later and still works as well.
Tax
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dtdavani

Participant

Posts: 12

Joined: Fri Sep 11, 2015 9:52 pm

Post Mon May 14, 2018 4:10 pm

Re: Indium chemistry

Hi Naeem,

Greg Chang in our facility conjugates anti-CD57 clone HCD57 to Indium which stains robustly ( MMI= 1000) at 1/800 dilution.

Best,
Dariush
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MarcelaAH

Participant

Posts: 6

Joined: Fri Jan 16, 2015 10:20 pm

Post Thu May 24, 2018 10:17 pm

Re: Indium chemistry

I just wanted to add my experience with the metals from Trace Sciences.

I mistakenly bought the two isotopes of Indium in the metallic form :? . I consulted with Mike and he suggested trying to dissolve with HCl concentrated. I read that the reaction of indium with HCl was slow so I added 15 uL every day until reaching 45uL and left the metal to dissolve for the weekend. After the metals completely dissolve in acid, I took it to 50mM in L buffer.

I noticed that when I put this metal with the polymer, the solution is a bit cloudy, but the labeling was successful. Fortunately this strategy worked for me and I am passing the advice in case anyone needs it.
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri May 25, 2018 12:07 am

Re: Indium chemistry

Thanks for the info Marcela!

Generally, when lanthanide solutions are cloudy, addition of a bit more acid clears them up. You might try adding aliquots of 3% nitric or something similar....usually doesn't take very much, so 5-10uL at a time should be sufficient.


Mike
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ReeceD

Participant

Posts: 2

Joined: Fri Jan 06, 2023 8:58 am

Post Wed Jun 28, 2023 10:09 am

Re: Indium chemistry

Hi CyTOF Forum,

Apologies for reviving a dead thread!
We want to expand the metals available to conjugate at the facility and are considering purchasing enriched Indium 115.
I was wondering if purchasing enriched indium in 10mg quantities from companies like Trace Sciences international still the best option for these isotopes?

Has anyone discovered suppliers which offer the metal in a format which is ideal for CyTOF purposes? It would be great to hear your experiences and any developments regarding conjugating metals not provided by SBIO

Thanks!

Reece
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