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Cadmium abs artefacts

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Giorgio

Participant

Posts: 6

Joined: Fri Sep 07, 2018 11:09 am

Post Mon Oct 17, 2022 4:17 pm

Cadmium abs artefacts

Hi all,
we are having some issues with Cadmium antibodies:
when we have more than one antibody in our cocktail we have either events along the diagonal, as if either antibodies clump or metals jump from one antibody to the other.
Did anybody experience anything like this?
Thanks,
Giorgio
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CRStevens

Master

Posts: 55

Joined: Thu Jul 17, 2014 5:07 pm

Post Mon Oct 17, 2022 4:32 pm

Re: Cadmium abs artefacts

Hey Giorgio,

If you dont mind explaining a little more. What kind of buffers are you using. Also I see in the filename "processed". Did you do any kind of cell processing to these samples?

The reason I ask is we had issues with the Cd antibodies with certain lyse/fix buffers previously and noticed the metals falling off, not necessarily jumping, tho.
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Giorgio

Participant

Posts: 6

Joined: Fri Sep 07, 2018 11:09 am

Post Wed Oct 19, 2022 7:50 am

Re: Cadmium abs artefacts

Hi,

processed is just the process of the fcs file for export form the instrument.
We see the same issue whether we do fixation or fix/perm, using aBioscience kit or Fluidigm kit.
Thanks,
Giorgio



CRStevens wrote:Hey Giorgio,

If you dont mind explaining a little more. What kind of buffers are you using. Also I see in the filename "processed". Did you do any kind of cell processing to these samples?

The reason I ask is we had issues with the Cd antibodies with certain lyse/fix buffers previously and noticed the metals falling off, not necessarily jumping, tho.
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mleipold

Guru

Posts: 4300

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Oct 19, 2022 4:37 pm

Re: Cadmium abs artefacts

Hi Giorgio,

What CyTOF model are you using? The *_Processed part isn't common.

Can you describe your wash steps after surface antibody staining? And can you explain what your panels are in each of the 3 figures? My understanding is that:
"CD3 CD56" has only CD3 and CD56, and does not contain CD8 (or maybe CD8 in a non-Cadium channel)
"CD8(6) CD8(12)" contains CD8 but not CD3 (or maybe CD3 in a non-Cadmium channel)
"CD3 CD8(6)" contains both CD3 and CD8 in Cadmium channels.

Is that correct? If so, insufficient washing and/or Ab titer might explain part of the diagonal you're point out.

One way you might be able to check: Gate your CD3+ cells, then plot CD4 vs CD8. If you have ion-exchange/scrambling, then I would expect a decent amount of CD4+CD8+ since the CD4+ would be falsely CD8+ due to scrambling. If this is instead isotopic impurity spillover (with no scrambling), then I would expect the CD4+ to remain CD8- and have little or no CD4+CD8+.

Also, NK cells are CD3- but some are CD8+. If your signal is coming from isotopic impurity-driven spillover (rather than metal ion exchange/scrambling), then those CD8+ NK cells should still have some "CD3"/110Cd signal and the CD8- NK cells shouldn't.


Mike

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