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Cadmium spillover

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AMitch

Participant

Posts: 19

Joined: Mon Nov 24, 2014 12:40 am

Post Thu May 28, 2020 2:48 pm

Cadmium spillover

I'm looking at tweaking a panel and want to shift some high expressing markers onto cadmium isotopes. Most of these should be mutually exclusive, but it would still be good to get an idea of the spillover into nearby channels - does anyone have any data on this?

Normally I'd just quickly run some diluted stock metal to find this out myself, but we're currently still locked out of the lab due the COVID-19 situation...
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roexx078

Participant

Posts: 3

Joined: Thu Feb 08, 2018 6:21 pm

Location: Nashville, TN, USA

Post Thu May 28, 2020 3:27 pm

Re: Cadmium spillover

I believe Fluidigm has some information at least on the relative purity of each cadmium lot- maybe in the product lit, or ask your FAS?

More practically, we recently moved all our major lineage markers to cadmium. Titrations last week looked excellent, essentially no spill channel to channel even at the highest antibody concentrations, and with antibodies that are usually a problem (looking at you, CD8). Even the brightest staining was about a log lower than we observe on the lanthinide channels, which in this case is an asset.
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desireeBCRT

Participant

Posts: 13

Joined: Mon Jan 06, 2014 11:56 am

Post Thu May 28, 2020 3:40 pm

Re: Cadmium spillover

Hi,
there is a spillovermatrix provided by Fluidigm. I hope the upload of the image worked.

Cd_Spillover.jpg


Best
Desiree
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mleipold

Guru

Posts: 2162

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu May 28, 2020 4:21 pm

Re: Cadmium spillover

Hi Andrew,

I have uploaded a PDF of histogram overlays for single CD45 labeled with Cd106/108/110/112/114/116 (I did not buy Cd113, since we are already using In113).

I also uploaded an Excel of Medians for each sample in each channel (both Raw MFI, and Normalized percentages) from the FlowJo analysis.


Mike
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mleipold

Guru

Posts: 2162

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu May 28, 2020 4:26 pm

Re: Cadmium spillover

And here's a similar experiment, with 3 different donors. CD45 barcodes were 6-choose-3. I stained them, then split into Single vs Combined Barcode samples.

Here are some PDFs, and an Excel output of MFI
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AMitch

Participant

Posts: 19

Joined: Mon Nov 24, 2014 12:40 am

Post Fri May 29, 2020 12:52 pm

Re: Cadmium spillover

Thanks all for the information and comments. As it turns out my local FAS saw the message as well and passed on the spillover matrix offline, but it's very helpful/reassuring to hear the first hand experience with the Cd isotopes and to see some hard data (Mike).

Cheers,

Andrew
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XMUCyTOF

Participant

Posts: 7

Joined: Tue Aug 07, 2018 3:09 am

Post Sun May 31, 2020 2:55 am

Re: Cadmium spillover

mleipold wrote:And here's a similar experiment, with 3 different donors. CD45 barcodes were 6-choose-3. I stained them, then split into Single vs Combined Barcode samples.

Here are some PDFs, and an Excel output of MFI


Hi, thank you for your first-hand information, it's very useful, and what's your conclusion? Especially the CD45 Barcoding, which is the best choice? I'm new here and not familiar with the spillover matrix :D

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