Non-specific extracellular staining despite FcR blocking
I am new to mass cytometry and currently experimenting with a 37-marker Tcell/NKcell oriented panel on a Helios cytometer. I work with fixated, cryopreserved human PBMCs that seem normal when I look at the major cell populations in flow cytometry (mono, lympho, B cells, NK).
In mass though, after cleaning the data and gating on T lymphocytes (CD45+CD3+), I am encountering an odd non-specifically stained cell population that's always about 3% of that gate. These cells display strange/uncommon phenotypes such as CD4+CD8+, CD16+CD8+, TCRgd+CD8+... and it goes on with about 15 antibodies. And when I gate one of these populations out, all of the other strange double positives disappear.
My staining protocol goes as follows (briefly, it's the Fluidigm protocol):
- aliquot 3 million cells (in PBS)
- block FcR (using Miltenyi blocking reagent for 10min at the recommended concentration)
- extracellular staining, then 2 wash steps
- fixation/permeabilization using Fluidigm Fix I and Perm S buffers, then 2 wash steps
- intracellular staining, then 2 wash steps
- fixation with freshly prepared 1.6% FA for 10min
- wash step then overnight iridium staining.
Has anyone been encountering similar problems using the Fluidigm protocol? Unfortunately I can't seem to figure out to attach any files or images to show what this strange staining looks like.
Thanks for your response!
Cheers, Carole