IgM for fixed blood

[lucrodn]

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

[rhannan]

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:


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

[lucrodn]

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