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Nanoparticles in CyTOF

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henkmei

Participant

Posts: 16

Joined: Wed Jul 30, 2014 9:44 pm

Post Mon Dec 07, 2015 10:05 pm

Re: Nanoparticles in CyTOF

Hi all,
I am not sure whether comparing signals elicited by a particle (or cell) suspended in water to signals coming from metal salts or nanoparticles dissolved in acid may work very well. There is certainly a correlation, but I doubt that one can serve well as a standard for quantifying the other.

We ran two assays to analyze metal loading of Ab (which could be extrapolated to metal content of Nanoparticle-conjugates provided that they completely dissolve). i) We used Ab capture beads incubated with known concentrations of metal labeled Ab and ii) dissolved the metal labeled Ab in tuning solution or other acid and running them in “solution mode”. I estimate the bead-based assay to be more practically meaningful for the application of the Ab conjugates, since signal intensities elicited by the Ab-conjugate-loaded beads comprise both, the metal loading of the Ab but also incorporate differences in ion transmission efficiency across the mass spectrum of the individual machine. The approach appears to work well, but does not permit the estimation of the number of metal atoms per Ab. In order to get these numbers, we ran additional ‘solution mode’ experiments.

I first diluted Ab conjugates and metal solutions (which I intended to use as reference with known concentration of metal ions) in water, aiming at being as close as possible to cell sample conditions (Ab conjugate in water). This gave me, as expected, detectable signals at varying levels for the conjugates but signals for metal salts were unexpectedly low for some, but not all metals, using data of tuning solution as a 2nd reference. I repeated this with new dilutions but similar results.
Based on that, I compared signals obtained from metal salts (and cisplatin) diluted in water versus spiked into tuning solution at identical concentrations. This revealed dramatic differences between water vs tuning (nitric acid), but also between metals (different salts or cisplatin). Attached figures (raw data, all panels represent the identical injection series, examples highlight the different "types" of metals/ metal behavior I noted in the whole experiment) show some of the measurements (CyTOF v2), successive injections are differently colored. No HF/’wash’ but water washes between injections.
In order to circumvent the differences which occur based on which metal is measured in which matrix, we ended up using tuning as reference for lanthanide labeled Abs, but separate SPEX Pt and Pd ICP-MS standard solutions for Pt and Pd labeled Abs, respectively. These standards were in 2%HCl, so we used 2% HCl as a matrix for Pt and Pd conjugates.

Possibly, at least some of the difference arose from different distribution of metal ions into liquid vs tubing depending on whether the liquid was water or acid.
Notably, lanthanide signals were comparably very low when salt solutions were diluted in water as opposed to tuning, while lanthanide-conjugate-labeled cells in water routinely give principally useful signals, so that chelated metal appear to show behavior differing from metal salt solutions. In line with the differential kinetics of tuning isotopes that can be observed when injection tuning solution, I also consider the potential retention of a given metal in the tubing to depend on the overall composition of metal salts in the injected solution.

I also want to note that Fluidigm provided on request a protocol that describes quantification of metal ions per Ab molecule, including an Excel spreadsheet which does the calculations. It is however limited to lanthanide metals which can be obtained through Fluidigm in MAXPAR kits, so it didn’t help us much for the platinum-labeled Abs.

Henrik
Attachments
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KalleB

Participant

Posts: 4

Joined: Tue Nov 24, 2015 10:41 am

Post Wed Dec 09, 2015 9:22 am

Re: Nanoparticles in CyTOF

Thanks for all your answers! =D

I don't quite understand the trick with the tuning solution, is it to get these transmission coefficients? (still reading up on this method so sorry for any stupid questions :P )

In my mind it would be better to run my standard/reference solutions of nanoparticles in water or buffer that resembles the cell environment?

Yes we mainly use gadolinium, I will keep in mind that you can/have to consider several isotopes. (We haven't run any experiments yet we just have access to one and we are looking into this and see if we can find a good use for it =) )
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mleipold

Guru

Posts: 2156

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Dec 09, 2015 4:23 pm

Re: Nanoparticles in CyTOF

Hi Kalle,

The purpose of diluting into the Tuning Solution (or one of the other ICP-MS standard solutions) is to be able to actually count your ions.

If you know the concentration of the ions in the standard solution (provided by the manufacturer, be it Fluidigm or someone else like Spex), then you can calculate the transmission coefficients for those metals that are similar in mass and chemistry to what is in your particular sample. Remember, there's a slight change in mass response (transmission efficiency) across the mass range, leading to slight changes in signal intensity for elements present at the same concentration. So, for example, the Tuning Solution contains 0.5 ppb each of Cs133 and Tm169; they're at the same concentration, but the Tm169 signal read by the CyTOF will be higher than the Cs133 signal.

Therefore, ideally, you want to use a reference metal that's close in mass to the one you're interested in, to minimize any variation in transmission coefficient. Once you know that, then you can calculate the number of metal ions in your particular analyte "unknown" solution.


Mike
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KalleB

Participant

Posts: 4

Joined: Tue Nov 24, 2015 10:41 am

Post Thu Dec 10, 2015 3:13 pm

Re: Nanoparticles in CyTOF

Ah okay. I'll have to read abit more about this I feel.

One question, do I use the tuning solution for both my reference with only nanoparticles and for my cells test? Since there could be a big difference measuring in water vs tuning solution according to the graphs above?

I'm very impressed how fast and good answers I get here, thanks alot once again :)

/Kalle
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mleipold

Guru

Posts: 2156

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Dec 10, 2015 4:13 pm

Re: Nanoparticles in CyTOF

Hi Kalle,

You can't use Tuning Solution to suspend your cells: the 2-3% nitric acid in the Tuning Solution would eat your cells.

I think the workflow would be to test the nanoparticles themselves in Tuning Solution to get a measure of their signal, then use that number as a reference in your cell experiment.


Mike
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anitamkant

Contributor

Posts: 46

Joined: Mon Nov 18, 2013 6:30 am

Post Thu Dec 10, 2015 8:37 pm

Re: Nanoparticles in CyTOF

Hi Kalle,
Thanks for initiating an interesting discussion on the topic of nanoparticles.
As per your post you are trying to measure the cellular uptake of nanoparticles of 5-10nm diameters consisting of different combinations of lanthanides.
Here are answers to your questions from our in house experts:
1) Will the nanoparticles be completely disintegrated in the ICP to single ions?

A: The plasma in the Helios/CyTOF system can completely vaporize and atomize metal nanoparticles up to 100 nm in size. Lanthanides are very efficiently ionized in the plasma.

2) Is there any risk of too high concentration of ions? in other words what is roughly the "highest concentration" recommended of a sample?

A: Yes. It would be possible to have enough nanoparticles in a cell that would yield so many ions that the detector would become saturated. The best way to determine this for your system is to incubate cells with different concentrations of nanoparticles and run from low to high concentrations. The cells should also be stained with Ir prior to running on the CyTOF.

3) What is the smartest way to correlate the signal to numbers of nanoparticles? Would it work to simply run a solution with known concentration of particles and use that as a reference?

A: To calculate number of nanoparticles per cell one needs to know
a) The transmission efficiency of one of the lanthanides from the nanoparticle;
b) Its signal per cell event as detected by Helios/CyTOF;
c) How many atoms of that lanthanide is present per particle (assuming composition of all the nanoparticles is uniform) as determined by an independent ICP-MS experiment
Answers to a and b will let one know how many atoms of the lanthanide were present on per cell basis. Combined with c one can determine the number of nanoparticles per cell.
To determine transmission efficiency of a lanthanide from the nanoparticle, one can run a standard solution of that lanthanide or if its atomic mass is close to the atomic mass of one of the isotopes in the tuning solution one can use the transmission efficiency of the isotope from the tuning solution. This needs to be determined empirically.

Please do not hesitate to contact your local Field Application Scientist for more in depth guidance and planning.

Regards,
Team Fluidigm
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