I want to stain PBMC such that I will be able to sort B cells that are IgE positive and sequence their genomes.
Does anybody of the fine scientists here have experience with this?
Particularly, do I have to stain he cells intra-cellularly or is it possible to stain them for IgE extra-cellularly?
And if intra-cellular is needed, will I be able to sequence them once they are fixed by the perm-fix procedure?
Thanks!
I don't have experience doing this but seems surface staining is enough. Maybe the protocol below can help.
Article A simple method for measuring human cell-bound IgE levels in...
Dear Gil,
I briefly wanted to message you, concerning your sort problem. To be honest I have not performed the exact same experiment, but working with B cells, I know it can be tricky to sort IgE+ plasma cells from peripheral blood of healthy human subjects and despite the abundance of IgE in serum, it's not trivial in atopic patients either. The attached publication from Horst et al. suggests to do a CD138 positive selection with MACS (from Miltenyi, which in my hands always worked great!) prior to the sort. They then stained for intracellular IgE and fixed with a formaldehyde based fix perm buffer (Miltenyi's Inside Fix) that will lead to nucleic acid fragmentation.
Article Detection and characterization of plasma cells in peripheral...
This will definitely interfere with your genomics application downstream and requires rather arduous optimization to get good sequencing results. Without knowing the exact nature of your experiment (transcriptome or genome sequencing) or the NGS sequencing capabilities from your core unit, I'd suggest that you use the above mentioned staining protocol and try it either with the commercially available reagents (and loads of optimization) or as some authors suggest with a methanol based fixative (that should allow your intracellular staining) and seems to give good results for NGS sequencing (see attached).
Article Cell fixation and preservation for droplet-based single-cell...
I hope that helps! Personally, I'd rather optimize the antibody stain (with methanol as fixative) as the sequencing protocol optimization after PFA fixation will a) take much longer until you have results and b) will be much more expensive in case it fails. I hope those considerations help!
All the best & good luck with your experiment,
Michael
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