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139La in panels

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It is fine to promote your company's reagents. Just make sure they are relevant to CyTOF, and do so in moderation and style :-)
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GregHopkins

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Joined: Tue Apr 11, 2017 8:39 pm

Location: 2seventy bio, Cambridge, MA

Post Wed Feb 23, 2022 2:15 pm

139La in panels

Hello everyone,

Its been years since I've had any antibodies conjugated to 139La in a panel, and with limited success. From what I remember, the conjugations were a crapshoot on yield and shelf life was generally short. Are there specific antibodies and reagents with which anyone here has had relative success and good track record of Ab performance?

Greg
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bbengsch

Participant

Posts: 4

Joined: Thu Jun 19, 2014 12:39 pm

Post Wed Feb 23, 2022 3:45 pm

Re: 139La in panels

We routinely use La139 for our MM-DOTA live/dead reagent, the stability that is problematic with ab conjugation as you also experienced doesnt seem to be a problem..
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MCOlivier

Contributor

Posts: 43

Joined: Mon Oct 05, 2015 9:48 am

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

Post Wed Feb 23, 2022 3:56 pm

Re: 139La in panels

Hi Greg,

I used once (twice, see beelow) the expedeon Lightning-Link La139 labelling kit for a large panel with 42 antibodies.

My first attempt was a fail, as the first lot I recieved was unproperly shipped (we discovered it staied a while at room temp during shippment) and did not work. But the second attemp with a new, properly shipped lot, gave good labelling efficiency of the AB. After staining, the metal did not leak to other ABs as no significant background in 139 channel was observed.

To my knowledge, Fluidigm retrieved 139La from their catalog because the metal did not stay captured in their polymer, and, if remember correctly, the metal was uptaken in the other polymers of the panel, even if already coupled to metals. I do not know what kind of polymer is used in the expedeon kits, but it seems the the metal is retained properly.
Hope this helps !
Best
Olivier
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GregHopkins

Contributor

Posts: 26

Joined: Tue Apr 11, 2017 8:39 pm

Location: 2seventy bio, Cambridge, MA

Post Wed Feb 23, 2022 4:40 pm

Re: 139La in panels

MCOlivier wrote:Hi Greg,

I used once (twice, see beelow) the expedeon Lightning-Link La139 labelling kit for a large panel with 42 antibodies.

My first attempt was a fail, as the first lot I recieved was unproperly shipped (we discovered it staied a while at room temp during shippment) and did not work. But the second attemp with a new, properly shipped lot, gave good labelling efficiency of the AB. After staining, the metal did not leak to other ABs as no significant background in 139 channel was observed.

To my knowledge, Fluidigm retrieved 139La from their catalog because the metal did not stay captured in their polymer, and, if remember correctly, the metal was uptaken in the other polymers of the panel, even if already coupled to metals. I do not know what kind of polymer is used in the expedeon kits, but it seems the the metal is retained properly.
Hope this helps !
Best
Olivier



Thanks Olivier. From your experience, it seems like the Expedeon kits might be worth a try. Is there any caveats about particular Ab characteristics (isotypes, etc) that would need to be taken into account with using the Expedeon kits? We have never tried them so I have minimal understanding of how they work (I've glanced at them in passing, but never looked too deeply into them).
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MCOlivier

Contributor

Posts: 43

Joined: Mon Oct 05, 2015 9:48 am

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

Post Wed Feb 23, 2022 4:49 pm

Re: 139La in panels

Dear Greg, here is the information I get quickly from one of the mails exchanges with the company in 2019 :

"Yes, our Lightning-Link Metal Labelling kits use amine chemistry to form a very stable covalent bond allowing the conjugate to be stable. As this is a new product we are still doing multiple stability testing on several metals and so far we have found they are stable up to 1 month when stored at -20⁰C with a cryoprotectant such as 50% glycerol. This is something we are still testing and believe could extend with time. This was tested on our antibody in-house so stability can vary depending on the antibody you use as the antibody tends to be the first to de-stabilise. I would recommend checking with the antibody supplier on your antibodies stability. "

I can't find out right now the specification sfor Isotypes specificity, but I imagine/guess that one-stranded Ig isotypes would be fine as for main covalent conjugation protocols, and that IgM would not work.
I also imagine they have more insight on caveats 3 years later :)

If I find back some more information, I will post it here :)

Best
Olivier
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CRStevens

Master

Posts: 55

Joined: Thu Jul 17, 2014 5:07 pm

Post Wed Feb 23, 2022 4:56 pm

Re: 139La in panels

I appreciate this discussion. I actually had missed that these reagents were available. My first thought is are they using a polymer chain such as the X8 and if not would the also decrease the staining index as you'd most likely have less metal? I'd be curious to try these and compare to the X8 as far as how they stain. Stability would obviously be a different question and concern.

-Chad
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JamesW

Contributor

Posts: 36

Joined: Tue Nov 21, 2017 6:59 am

Post Thu Feb 24, 2022 1:24 am

Re: 139La in panels

Hello everyone,

Actually are these products still available? At least 139La seems missing from the current list of lighting link kits and after some searching I am only finding dead links and "Product discontinued"

Lightning-Link® 139La Antibody Labeling Kit
Catalog Number:M139-0010
Availability: Product Discontinued

My record on X8 polymer 139La conjugation is two attempts and two failures. Barely detectable staining, antibody was anti-human CD45 clone HI30. So keen to try another method.

Best,
James
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mleipold

Guru

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Location: Stanford HIMC, CA, USA

Post Thu Feb 24, 2022 10:32 pm

Re: 139La in panels

Hi Chad,

I don't have any direct info on what is in the Lightning Link kit; I don't think I asked when I talked with them at a CYTO a few years ago, and I couldn't find anything clear on their website.

Olivier's answer from the vendor and the Expedeon/Abcam protocol state that you need to use buffers without amines. Therefore, my *guess* is that it's something like NHS-DOTA (eg: https://www.macrocyclics.com/online-cat ... nhs-ester/) pre-loaded with the metal of interest and then lyophilized for stability (since NHS-esters will react with water and therefore prevent reaction with your antibody).

If that is the case, then you'd want to use it with something fairly abundant (eg, CD45), since it would be a one-metal-per-chelate ratio like the ITCB-EDTA-Pd is.


Olivier: when you were successful with the Lightning Link, what marker did you label?


Mike
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MCOlivier

Contributor

Posts: 43

Joined: Mon Oct 05, 2015 9:48 am

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

Post Fri Feb 25, 2022 11:29 am

Re: 139La in panels

Hi Mike,
And thx for this chemistry lesson ;-) !

"Sadly", I choose Perforin, which is very abundant/well stained in stimulated cells, but in the protocol I used, we could not stimulate the cells, then, staining is very very low... However, AB captured on beads to control for conjugation efficiency was totally fine !

To summarize, "bad target/metal/no stimulation choice" I would say, but this was a bit like the cherry on the cake, so we still tried

Best
Olivier
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GregHopkins

Contributor

Posts: 26

Joined: Tue Apr 11, 2017 8:39 pm

Location: 2seventy bio, Cambridge, MA

Post Wed Jun 08, 2022 4:34 pm

Re: 139La in panels

In the absence of the Expedeon kits, are others, what sources for metal were those who conjugated it via X8 using? Was it something from Trace or Sigma?

I must say I am a bit worried about nthe metals not being retained, but it may be worth a trial with a high expression target with biomodal or bimodal-esque distribution. I've never really looked into the oxidative spill from this channel but I imagine it might be pretty noticeable.
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