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Non-specific binding of Intracellular antibodies - Help!

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tantalod

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Posts: 2

Joined: Tue Nov 14, 2017 3:37 am

Post Wed Nov 15, 2017 2:12 am

Non-specific binding of Intracellular antibodies - Help!

Hey All, Long time reader - first time poster

I’ve run into some issues with my intracellular CyTOF staining. Seems like we get a lot of non-specific binding from our intracellular antibodies such as IL2/Eomes, which we would expect to find in T cells, not in CD33+ cells (see attached photo ). Our extracellular markers as a whole seem to correlate with what we would expect.

Attached is a file with some ViSNE plots showing this non-specific binding. The file has two panels from two experiments:

1) Melanoma sample (Top Panel)
2) PBMC (bottom Panel)

1) ViSNE plots showing expression of 5 intracellular markers and CD33 on Melanoma samples which were stimulated with PMA/Iono for 5 hours and then stained for a variety of extracellular and intracellular markers, many T cells markers.
My staining protocol included:
- A 30min block with 10% Human AB serum
- Extracellular antibody stain
- Fix/Perm using FOXP3 transcription factor kit from eBioscience (2 perm wash steps before intracellular stain).
- intracellular antibody stain (made up in perm wash from FOXp3 kit)
- Two permwashes
- Overnight Iridium in 4% PFA

2) The panel below shows ViSNE plots from an experiment using PBMCs. This experiment was completed using the same antibodies, same staining procedure as the above.

Has anyone else run into this issue? If so, how have you over come this?
Any help would be greatly appreciated!
Daniela


Image
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mleipold

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Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Nov 15, 2017 4:50 pm

Re: Non-specific binding of Intracellular antibodies - Help!

Hi Daniela,

You might try heparin treatment, to see if that helps:
"Heparin reduces nonspecific eosinophil staining artifacts in mass cytometry experiments"
Adeeb H. Rahman, Leticia Tordesillas and M. Cecilia Berin
Cytometry A, 2016
http://dx.doi.org/10.1002/cyto.a.22826


Mike
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carynv

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Posts: 5

Joined: Tue Feb 14, 2017 12:36 am

Post Wed Nov 15, 2017 10:42 pm

Re: Non-specific binding of Intracellular antibodies - Help!

Hey Daniela!

We see significant non-specific labelling of intracellular Abs (especially the pSTATs) when using smart tube buffer cells. Heparin treatment (following the paper Mike referenced) has been the only blocking step that solves this but needs to be done after permeabilisation, whether it be methanol perm or eBioscience fix/perm.
I have done tons of troubleshooting with this and happy to send you the results if you want more info :D

Caryn
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tantalod

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Posts: 2

Joined: Tue Nov 14, 2017 3:37 am

Post Mon Nov 20, 2017 10:20 pm

Re: Non-specific binding of Intracellular antibodies - Help!

Thanks guys! Ill try this out :)
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newmmat

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Posts: 1

Joined: Fri Dec 18, 2020 3:52 am

Post Tue May 11, 2021 9:19 pm

Re: Non-specific binding of Intracellular antibodies - Help!

Hi all,

First time poster - I know this post was from a while ago, but hoping to get some insight for some upcoming experiments regarding heparin for elimination of non-specific signal for intracellular stains. Specifically, I'm going to be looking at phospho signaling (including pSTAT5) in peripheral blood samples that have been banked in Smart Buffer. These will also be stained for surface markers of interest.

In regards to the paper (Rahman et al, 2016), it seems that heparin was used prior to surface stains/permeabilization - potentially in place of Fc block? It seems that some who have tried heparin blocking specifically with samples frozen in smart buffer say that this should be done after permeabilization with methanol (following the phospho-stain protocol). If possible, I was hoping to get some more insight on the specific steps used for heparin blocking in smart buffer samples, and whether or not Fc block should still be used in prior steps to further attenuate non-specific binding.

Many thanks!

- Matt
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mleipold

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Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu May 13, 2021 4:42 pm

Re: Non-specific binding of Intracellular antibodies - Help!

HI Matt,

Fc block and heparin do different things, so you can use both.

In a more recent paper from Mt Sinai (https://doi.org/10.1002/cyto.a.24317), they state:

"In experiments to evaluate the performance of additional antibody clones (Table 2) pre‐ and post‐Prot1 fixation, replicate aliquots of the same blood samples were either stained as fresh whole blood and then fixed and frozen or were fixed and frozen as unstained aliquots and then thawed and lysed as described above. The thawed samples were then resuspended in a volume of Cell Staining Buffer equal to the volume of the original blood aliquot for staining. SmartTube fixation and thaw/lyse results in partial permeabilization of cells and, consistent with prior results,6 we found that addition of 100 U/ml of heparin was critical to prevent non‐specific staining of eosinophils when adding antibodies to SmartTube‐fixed whole blood. However, this was not found to be necessary when staining fresh whole blood. "

So, here, they didn't do additional methanol perm, just "used" the "leftover" perm from the SmartTube PROT1 fixation. In other words, whether you use heparin is not dependent upon methanol.


Mike

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