Indium chemistry

[ssivajothi]

Hi all,

Thank you for all this information. I am starting to look beyond Fluidigm supplied metals to expand my panels. (This is important for the imaging mass cytometry application right now as there is no 'compensation' or 'gating out' of signal overlap. Any signal spillover appears as a shadow image in the channel being spilled into; looking to either ends of the spectrum will help add two or three more markers).

I have read almost all the threads on custom conjugations here and have a very beginner question: how do you find the isotope mass of a metal that is being purchased from Sigma? For example, Indium chloride- is it 115 or 113? Sorry if this is really stupid!

Thank you,
Santhosh

[MCOlivier]

Hi Santhosh,

Not that stupid, don't worry (I asked the same ^^).

You usually have to call the supplier and ask for detailed metal analysis of the lot they are curently selling. There you will find the specification of the contaminents that are succeptible to be present in the "powder", and specially for Indium, the ratio of 113/115. As I remember, Sigma do not enrich 113 or 115 isotope, so they should be "naturally" abundant, ie 95.7% 115 vs 4.3% 113.

Hope I'm clear.

Regards

[bboden]

Dear Santhosh,

We faced the same issues in regards to signal spillover in imaging mass cytometry. We have thus developed compensation for suspension and imaging mass cytometry. You can find our manuscript here https://www.biorxiv.org/content/early/2017/09/07/185744
Soon the final version of the manuscript will be published as well.

Best,
Bernd

[mleipold]

Hi Santhosh,

To follow up further: unless specifically stated by the supplier (Trace Sciences, Cambridge Isotopes, etc), you should assume that any metal salt you buy is of natural abundance isotopic distribution. Technically, the exact isotopic distribution can vary by source (the exact mine they got the ore from, etc). But to a very good first approximation, that can be ignored.

Here are a few links to tables of natural abundance isotopic distributions for the elements: viewtopic.php?f=9&t=11

Reminder: isotopic purity is not the same as elemental purity. So, for example, Sigma sells several Indium (III) chlorides, ranging from 97% (334073) to >99.999% (429414, trace metals basis). The 97% would have up to 3% non-Indium, while the >99.999% would have no more than 0.001% non-Indium. However, *both* would be ~96% In115, 4% In113 (so, *not* hugely useful to label an antibody, but probably OK for a live-dead)

From any supplier, the exact lot that you get usually comes with a spec sheet detailing at least some of the non-M impurities.....the higher the purity, the longer the spec sheet, usually.


Mike

[mleipold]

Hi Jahangir,

I don't use the Fluidigm BC reagents for barcoding (I use Pd-CD45 or other antibodies, when I do BC). But Indium is definitely compatible with Palladium in the same panel, so it's not something related to spillover.

I'm sure other people (Greg, Adeeb, etc) will chime in about using Indium and Fluidigm or Zunder-like small molecule BCs containing Pd.


Mike

[ErinSimonds]

Hi Jahangir,

I routinely use the Fluidigm 20plex Pd barcode kit alongside indium-tagged antibodies, Y89-tagged antibodies, Ce140-tagged antibodies, Bi209-tagged antibodies, cisplatin viability staining, and iridium DNA staining. This combo has worked well for me across a wide variety of tumor/immune tissues and cell types. I don’t use Rh103, but that should work too. There is nothing in the Pd chemistry or isotopic impurities that should prevent you from using them alongside indium-113 or 115. Just be sure to put 113 and 115 on more abundant targets (CD3 or similar) because they are not as bright as the lanthanides.

Could you please relay to the Fluidigm Tech you spoke with that this combination works? I’d hate for more users to be discouraged from trying it.

Erin

[ssivajothi]

Hi all,

@Olivier- thank you for the info, yes this clears up my question and it is reassuring that I wasn't alone in wondering about this!

@Bernd- yes, I saw your paper on Biorxiv and I spoke to Vito about the details of preparing the metal coated compensation slide. He mentioned the detailed protocol will be published with the final paper so I am eagerly awaiting it.

@mike- as always thank you for the detailed info, it does clear up a lot of things and provides some food for thought.
1) When you say the In is OK for live-dead but not for labeling to antibody, can you explain why? If I am using only the 115 and leaving the 113 channel empty is it then okay to label with antibody? Or is there some other reason for this statement?
2) I see that Trace sells most Pd isotopes (104, 105, 106, 108, 110) at >96% purity. Is it possible to use all these channels to tag and detect different markers?

Thanks again,
Santhosh

[mleipold]

Hi Santhosh,

If you are using Nat Abund Indium, that's ~96% In115 and ~4% In113. Therefore, you'll have In113 signal at about 4% of your In115 signal.

In the case of a live-dead, you're taking the Dead-neg events forward as Live Intact Singlets. Therefore, your In113 spillover from the In115 would be small (4% of a small number is an even smaller number, and should be background).

One of my current panels has LiveDead on In-natabund (monitor the 115 channel), and CD57 on the In113 channel (from Trace; ~93% In113, ~7% In115 according to the spec sheet). In this case, I actually have a minor but detectable spill from the CD57 into the In115 (Livedead) channel, which shows up as Dead-mid. I've done the backgating (looking at In115 signal on CD57+ and CD57-) to convince myself that things are resolved. Additionally, I still have 0.5-1log separation between the Dead-mid (CD57 spillover) and the Dead+ (genuinely dead), so I don't worry about it.

(I made a big stock of the nat abund Indium DOTA-maleimide years ago, before we got the pure isotopes from Trace....that's why I haven't moved things).


Regarding the Trace Pd stocks: yes, you can use them, particularly with the small-molecule (Zunder-like) BC agents. However, if you make antibody BC (eg, CD45), be sure to look at your spillovers, particularly into the 105 channel...if I recall, the 104 and 106 both have noticeable 105 spills, which can complicate using them with 105. My current BC project is small enough that I'm avoiding the problem by using this 2-of-5 scheme: 89/102/105/108/110


Mike

[ssivajothi]

Hi Mike,

Thanks for the clarifications.

Regarding the Pd isotopes, do you by any chance have some kind of spillover matrix? Are 104 and 106 the major culprits? Would it be safe to use 102, 105, 108 and 110 without spillover worry?

Since I plan to use the Pd channel for regular markers (not for barcoding), I am assuming I should use the Mei protocol to conjugate Pd to antibody?

Thanks again,
Santhosh

[Jahangir]

Dear Mike and Erin,

Many thanks for your replies. I feel much more confident now and I will purchase purified Indium-115 and 113 metal salts from Trace Sciences, as the Indium (III) Chloride isn't isotopically enriched (to my knowledge).

I have another question, Erin, you mentioned the Rh103 metal. To my knowledge, is that used as a DNA intercalator? If so, is that any better than the 191/193 Iridium intercalators? Also, has anyone tried to conjugate Rh103 to DTPA chelators for antibody conjugation?

Many thanks again for all responses,

Jahangir