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Alternative sources for isotopes

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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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GregBehbehani

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Location: The Ohio State University, Columbus, Ohio

Post Wed May 04, 2016 11:34 pm

Re: Alternative sources for isotopes

I can agree with everything Mike has posted.

I also buy Gd157 from Trace and my spec sheet is identical. When run on a Helios, an antibody labeled with 157 (same BD capture beads) gives ~6% spillover into Gd158, this is why Fluidigm doesn't sell Gd157. I don't see any signficiant signal in any non-Gd channel.

I think Trace bought most (or all) of their isotopically purified metals in bulk and just converts the oxide into whatever form you requested (i.e. GdCl3) to fill your order. Thus, the relative isotopic purity is always the same. I can't see any reason they couldn't make Gd157 more pure, but 92% Gd157 with 5.4% Gd158 is apparently what we're stuck with. Plan accordingly.

The old CyTOF 1 amplifier gets suppressed for ~100ns after a large number of ions come through; Mike, this is most likely why you see less Gd158 spillover than you would expect. If you repeat the same bead capture experiment on a CyTOF 2 or Helios, I expect you'd see the expected 5-6% spillover.


Greg
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Jahangir

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Posts: 37

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Post Thu Jan 18, 2018 10:30 am

Re: Alternative sources for isotopes

Hi Mike,

You mentioned in your earlier post on Tue May 03, 2016 about sourcing the chloride version of Gd157 and then dissolving it in something and loading it in.

Could you explain this a bit further please? What would you dissolve chloride metals in? (Would it be in the L buffer possibly) And would you then carry on with the antibody conjugation as normal?

Many thanks,

Jahangir
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AdeebR

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Location: NYC

Post Thu Jan 18, 2018 3:17 pm

Re: Alternative sources for isotopes

Hi Jahangir,

Most lanthanide salts are pretty water soluble. I've typically dissolved lanthanide salts in either H20 or 0.01M HCL to prepare a 2M stock solution, and then dilute this to 50mM working solution in L buffer.

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

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Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Thu Jan 18, 2018 3:37 pm

Re: Alternative sources for isotopes

Hi Jahangir,

I would second what Adeeb has posted.

Most of the salts will dissolve fine in L-Buffer. For those that won't, typically a bit of acid (HCl or HNO3) will solubilize them. I used to worry about this acid, but after the recent publication by Han et al. (Cytometry A, Dec. 2017), we've successfully loaded polymer at relatively high acid concentrations (5% HNO3), so if you need a bit of acid it shouldn't hurt anything. You can basically then perform the conjugation as normal, though if you use a really high acid concentration (as in the Han publication) some extra washes to remove the acid before conjugation would probably be helpful.

best of luck,

Greg
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Jahangir

Contributor

Posts: 37

Joined: Sun Oct 29, 2017 6:34 pm

Post Thu Jan 18, 2018 3:51 pm

Re: Alternative sources for isotopes

Many thanks for your replies Adeeb and Greg. I have a few more questions if you don't mind.

- When purchasing heavy metals from companies such as Sigma, does it matter if the metals are in the form of a monochloride or trichloride? Would this affect the conjugation process in any way?

- Also sometimes these metal salts also have water molecules bound to them and they're in the form of a salt hydrate, such as Dysprosium(III) chloride versus Dysprosium(III) chloride hexahydrate (both found here - https://www.sigmaaldrich.com/catalog/se ... us=product). Does the latter affect the conjugation process in any way? And which is more preferable for conjugating?

- Does it matter which salt is bound to the metal? I.e. if you buy it in the form of a fluoride, iodide, bromide, nitrate, acetate, chloride, sulfide, etc, would any of these have an affect on the conjugation process? (I know that metals in the oxide form won't work at all in the conjugation process). And which is the most preferable/optimal form when buying metals to conjugate antibodies to?

- And if you're buying metals in the form of a nitrate, does that mean you would have to dissolve it in a similar acid (such as nitric acid for nitrates or hydrochloric acid for chloride) or can you simply dissolve this is MaxPar water also?

Many thanks again for all your help,

Jahangir
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mleipold

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Location: Stanford HIMC, CA, USA

Post Thu Jan 18, 2018 4:07 pm

Re: Alternative sources for isotopes

Hi Jahangir,

Almost all nitrate salts are water-soluble. Many/most chloride salts are also water-soluble, but not all (eg, AgCl has a solubility constant in the 1e-14 molar range, I think). Many fluorides and sulfides are *not* water-soluble. Some acetates are, some aren't. You can get a lot of info on this from any basic chemistry solubility table (looking at both the cation and the anion).

Waters of hydration will not affect conjugation. You'll just have to take their weight into account when calculating how many mg to dissolve to get the appropriate stock solution.

Monochloride vs trichloride would imply that the metal can be found in more than one oxidation state (+1 vs +3). Usually, one oxidation state is more water-soluble than others (eg, Fe2+ vs Fe3+), which again can usually be found in the chemistry solubility tables.


I wouldn't worry too much about dissolving a nitrate with HCl or a chloride with nitric acid; the pH is the most important thing in solubility, and once it's in your chelator, it is "solubilized". The only time this would make a difference are those edge cases like silver, where the nitrate is highly soluble and the chloride is highly *INSOLUBLE*. As I said, nitrates are almost always water-soluble, so if you have to choose one acid, I'd probably stick with nitric.



Mike
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GregBehbehani

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Posts: 80

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Thu Jan 18, 2018 4:19 pm

Re: Alternative sources for isotopes

Hi Jahangir,

My answers below.

best of luck,

Greg

- When purchasing heavy metals from companies such as Sigma, does it matter if the metals are in the form of a monochloride or trichloride? Would this affect the conjugation process in any way?
**Yes, it matters quite a bit. You need to buy a salt that will disassociate into a 3+ metal ion for optimal chelation. Most of the lanthanides only form 3+ cations, but a few salts are in other oxidation states and will not chelate correctly 4+ ions are typically fine, but 2+ or 1+ cations often will not stay chelated.

- Also sometimes these metal salts also have water molecules bound to them and they're in the form of a salt hydrate, such as Dysprosium(III) chloride versus Dysprosium(III) chloride hexahydrate (both found here - https://www.sigmaaldrich.com/catalog/se ... us=product). Does the latter affect the conjugation process in any way? And which is more preferable for conjugating?
**You'll be dissolving the salts in water, so it won't matter if you start with a hydrate as long as the salt is in the correct oxidation state (i.e. (III)), and as Mike points out, you use the correct molar amount of metal. Typically, the hydrate is more water-soluble and thus a better choice. I'm not sure why you would buy Dysporsium from Sigma though; it will be a mixture of 7 isotopic states: 0.056% 156Dy; 0.095% 158Dy; 2.329% 160Dy; 18.889% 161Dy; 25.475% 162Dy; 24.896% 163Dy; 28.260% 164Dy.

- Does it matter which salt is bound to the metal? I.e. if you buy it in the form of a fluoride, iodide, bromide, nitrate, acetate, chloride, sulfide, etc, would any of these have an affect on the conjugation process? (I know that metals in the oxide form won't work at all in the conjugation process). And which is the most preferable/optimal form when buying metals to conjugate antibodies to?
**I'd agree with what Mike just posted on this. What matters is the oxidation state and solubility; you need the salt to be water soluble. The associated anion doesn't matter much and will be washed away in the buffer exchange after polymer loading.

- And if you're buying metals in the form of a nitrate, does that mean you would have to dissolve it in a similar acid (such as nitric acid for nitrates or hydrochloric acid for chloride) or can you simply dissolve this is MaxPar water also?
** If the metal salt dissolves in L-Buffer or water, you don't need any acid at all. In the unusual cases where acid is needed, we've dissolved in both HNO3 and HCl, but I would agree with Mike's point about rare cases where the nitrate is soluble and the chloride isn't, so nitric acid might be a safer bet.
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mleipold

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Location: Stanford HIMC, CA, USA

Post Thu Jan 18, 2018 4:27 pm

Re: Alternative sources for isotopes

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mleipold

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Posts: 2095

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Jan 18, 2018 5:05 pm

Re: Alternative sources for isotopes

One followup to Greg's point: for long-term storage, the salts are more stable if kept in slightly acidic conditions. L-buffer should be fine, but if you're using just MilliQ, you probably want to add a bit of acid. Otherwise, over time (months to years), the lanthanides can sometimes form oxides, which precipitate.

You don't need much: I think I was adding 100uL of 3% nitric to 10mL of MilliQ.
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JamesW

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Posts: 26

Joined: Tue Nov 21, 2017 6:59 am

Post Fri Jan 19, 2018 12:45 am

Re: Alternative sources for isotopes

Hello,
Sorry to jump in on a topic but since this thread is active I wanted to take the opportunity to ask approximately how much people were being quoted for metal salts from Trace sciences.

I recently obtained a quote for Indium and Palladium Isotopes from the Japanese distributor for Trace and they came in at an eye watering average of 300000 yen (about 2700 USD) for 10mg, which is the minimum they sell. That seems fairly out of line with some earlier info from mike which suggested something closer to 650 USD (Post Fri Jan 24, 2014). The purity requested was the highest they had (between 93-99%Atom).

Often things are unreasonably expensive in Japan but I did want to know if the distributor is inflating the price unreasonably.

Thanks,
James Wing
IFReC, Osaka University
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