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IgM for fixed blood

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lucrodn

Participant

Posts: 2

Joined: Mon May 27, 2024 9:27 am

Post Mon Jun 03, 2024 2:03 pm

IgM for fixed blood

Hi everyone,

We are trying to design an Ab panel to stain peripheral blood samples fixed with Proteomic Stabilizer. When titrating IgM (clone MHM-88), we could observe a high expression on T cells, most likely due to fixation.

I would like to ask if anyone has used a different IgM clone that works well with fixed blood.

Thanks in advance!

Lucía
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rhannan

Participant

Posts: 11

Joined: Tue Feb 15, 2022 5:05 pm

Post Tue Jun 04, 2024 3:57 pm

Re: IgM for fixed blood

Lucía,

I don't have experience with other clones of IgM ab, so feel free to stop reading here as this won't be an explicit answer to your question.


We've had success using MHM-88 post-fix staining. It's certainly dimmer in post-fix for an equivalent titer, but we haven't had T cells show up as highly positive. IgM/D plots for T and B Cells from whole blood/whole blood post-fix/and regular live PBMCs:
Image

Are your IgMhi T Cells as bright as IgM+ CD19+ cells? Are other cell populations? It could be a case of increased overall background. We've had that problem with other Ig staining (IgG/IgA) in post-smarttube samples, and I chalk that up to the cells being fixed in situ in human blood - with all the endogenous plasma Ig's being fixed right onto the surface of the cells.

Could you share your collection+staining protocol? Wonder where the differences lie.

Riley
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lucrodn

Participant

Posts: 2

Joined: Mon May 27, 2024 9:27 am

Post Thu Jul 18, 2024 4:31 pm

Re: IgM for fixed blood

Hi Riley,

Thanks for your reply!

We work with whole blood, stain with cisPt and fix with Proteomic Stabilizer. After thawing, RBC are lysed. Then, we block with heparin + Fc block and we do surface staining. Finally, we fix with PFA and stain with Ir.

Find attached the plots on B cells, T cells and granulocytes, respectively. Although median intensity is higher on B cells, maximum intensity is similar in all populations.

Thanks again,
Lucía
Attachments
pastedImage.png
IgM expression on B cells, T cells and granulocytes, respectively
pastedImage.png (295.64 KiB) Viewed 59 times

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