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Seeking Experience with Cadmium Conjugates

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annakus

Participant

Posts: 1

Joined: Wed Apr 01, 2020 6:00 pm

Post Fri Apr 03, 2020 3:48 pm

Seeking Experience with Cadmium Conjugates

Hi All,

I'm working on developing a new CyTOF panel and am hoping to take full advantage of the new Cadmium metals to expand the number of targets I can include in my panel. In the past, I've successfully done live cell barcoding with cadmium conjugated antibodies using both b2m and CD45 (separately) without issue, and have also used CD19-, CD3-, and CD14-Cd conjugates with varying results. CD19-Cd113 worked quite well at a very low titer (0.2uL), while CD3-Cd116 and CD14-Cd114 were much "dimmer" than I'm accustomed to even at higher titers, although still discernible. Knowing that I'll likely lose resolution in signal for targets conjugated to Cadmium metals rather than metals from the typical 141-176 range, I'm wondering how feasible it is to use the Cadmiums for purposes apart from barcoding or defining very broad cell lineages.

So my question is, which antibody targets have you all used successfully with the Cadmiums? Would targets like CD4 or CD8 be bright/abundant enough to use? Have you experienced differing labeling efficiency between the different Cd metals?

For reference, this will be a mouse panel and will include a PMA/I stim that may result in some downregulation of surface markers.

I super appreciate y'all sharing your experience and insights with me!

Anna
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MCOlivier

Contributor

Posts: 32

Joined: Mon Oct 05, 2015 9:48 am

Location: European Genomic Institute for Diabetes, Universitity of Lille, Institut Pasteur de Lille, France

Post Mon Apr 06, 2020 8:51 am

Re: Seeking Experience with Cadmium Conjugates

Hi Anna,

I've conjugated CD8 with 116Cd in human and got very clean, discriminative stainings.
I really think that highly expressed markers such as CD4/8/Ly6G/CD19.... should give good results.

Best regards ;-)
Olivier
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JamesW

Contributor

Posts: 26

Joined: Tue Nov 21, 2017 6:59 am

Post Tue Apr 07, 2020 2:26 am

Re: Seeking Experience with Cadmium Conjugates

Dear Anna,

So far we have tested mouse 111 CD8, 112 TCRb, 114 CD3, 116 B220 and Human 110 CD3, 111 CD4, 112 CD8 and 114 CD14. My overall impression is that they are a little dim but certainly workable for lineage markers. Of those only the 114Cd murine CD3 was giving what I considered poor discrimination, it was staining but the separation was not great. That was actually a second try after we did the same clone (Purified anti-mouse CD3ε Antibody (145-2C11)) with 111Cd and which gave almost no staining.

I should mention that I was doing all of this with a protocol including saponin based intracellular staining (eBio Foxp3 buffer set, used after the lineage staining) which is reported to generate some possible problems with cadmium reagents. I pretty much always use intracellular staining so have not tested without, so cant say if things would look nicer without. I used the Cadmiums alongside 113 and 115 indium and they seem to fit well together without major crosschannel problems.

The other point worth mentioning is that our average recovery for lanatanide/indium/cisplatin labelled antibodies is about 70-90% whereas for cadmium it is more like 30-50%

Hope that is helpful.
Best,
James
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dtelad11

Master

Posts: 112

Joined: Mon Oct 31, 2016 6:26 pm

Post Wed Apr 29, 2020 1:00 pm

Re: Seeking Experience with Cadmium Conjugates

James, do you have any data you could share in this context? (FCS files with Cadmium + comparison to FCS files with same antibodies on non-Cadmium channels, for example?)
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JamesW

Contributor

Posts: 26

Joined: Tue Nov 21, 2017 6:59 am

Post Thu May 21, 2020 9:00 am

Re: Seeking Experience with Cadmium Conjugates

Hi El-ad,

Sorry missed your reply at first. Here is an example of some of the new Cd stains vs some of my previous antibodies. Its not the same sample.

Best,
James
Gating example.pdf
(303.92 KiB) Downloaded 36 times

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