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148 spillover into 164

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nkhanbham

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Posts: 53

Joined: Wed Feb 25, 2015 3:03 pm

Post Fri Mar 02, 2018 2:50 pm

148 spillover into 164

I recently noticed that I get a lot of spill from the 148 channel into 164, explained by oxidation. Looking back, through several experiments I see this is a consistent occurrence. I dont see it with other channels. Is this a problem that others have also found with 148/164? I am going to titrate down my antibody on 148Nd channel and hope i |can reduce the spill into 164Dy and still get a decent 148 signal.
Naeem
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mleipold

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Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Mar 02, 2018 3:57 pm

Re: 148 spillover into 164

Hi Naeem,

What marker do you have on 148? What is its signal intensity?

The reason I ask: there are a number of Nd isotopes, and yes, some of them that you get from Fluidigm are "cleaner" than others, but that is an issue of isotopic purity (eg, Nd146 in your Nd145 stock). However, no one Nd isotope oxidizes more or less than the other Nd isotopes: on a per atom basis, Nd148 and Nd142 should oxidize the same amount.

However, any sort of spillover, including oxide/M+16, is proportional to signal intensity at mass M. Therefore, if you have a really abundant marker at M (eg, CD45, CD57, etc) with a signal intensity into the high 100s-mid 1000s Dual, then you'll have a lot more spill in M+16. But that's a panel design issue, not something inherent to Nd148 specifically.


Mike
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nkhanbham

Master

Posts: 53

Joined: Wed Feb 25, 2015 3:03 pm

Post Fri Mar 02, 2018 5:08 pm

Re: 148 spillover into 164

I have CD34 on 148Nd, and it gives high intensity staining (high 100s-1000+). I think I will have to use less.
Thanks Mike
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nkhanbham

Master

Posts: 53

Joined: Wed Feb 25, 2015 3:03 pm

Post Fri Mar 09, 2018 8:03 am

Re: 148 spillover into 164

There is probably a logical answer to this next question. Why does an oxidised 148Nd particle still give a signal in the 148 channel? Surely if its now atomic mass 164, it only gets detected at 164.
Naeem
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ChristophS

Contributor

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Joined: Thu May 07, 2015 2:11 pm

Location: Switzerland

Post Fri Mar 09, 2018 8:55 am

Re: 148 spillover into 164

Hi Naeem,

not all the metal will be oxidized. Supposedly the way the instrument is tuned it should be always less than 3% (and that is for 139La which apparently gets oxidized the most readily among the Lanthanides).
Therefore a signal in a +16 channel will always be a fraction (in the 1/100th range) of the originator channel. Only if a signal there is in the several hundreds it will disturb the receiving channel (and that´s why you want to titer that originator signal down).

Christoph
Christoph Schwärzler
Director Cytometry
Flow Cytometry Facility (https://www.cytometry.uzh.ch/en.html)
University Zürich

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