Wed Jan 24, 2018 11:54 pm by GregBehbehani
Hi Sara,
I would echo Mike's comments; it's certainly possible to do this, but it will all depend on the specific antigens and antibodies you're studying.
Most of the original phospho-flow studies done in the Nolan lab were performed with a single post-methanol staining step, but this greatly limited the surface markers that could be studied to those that were stable after methanol (in my experience, only about 50% of biologically relevant surface proteins). If you're perm is saponin, then you'll fare much better (most surface proteins are fine after saponin), but just be aware of the recall that Fluidigm had on some of their buffers, as a small subset of Fluidigm antibodies are labeled with a different polymer (other than X8) which can non-specifically bind to some lots of saponin. You should also be aware that some surface proteins have an intracellular pool that can cause post-perm staining to be brighter than pre-perm (and potentially change immunophenotype assignments).
Fortunately, if you've already got a protocol that works by regular flow with a single staining step, then you should be fine to just convert this to a CyTOF experiment (same fix/perm, just substitute metal labeled versions of the same antibody clones you use). Otherwise, as Mike suggests, you'll have to validate the optimal conditions under which your antibodies bind (if this isn't already published). Fortunately, without having to worry too much about compensation, you can quickly test your whole panel under a variety of fix/perm conditions and you should be able to sort this out fairly quickly.
Best of luck,
Greg