I need to detect and measure the abundance of cytoplasmatic proteins (AIM2, GBP1, GBP4) and nucleic proteins (IFI16), all apparently soluble, using direct detection by flow cytometry with fluorochrome-conjugated antibodies in human mo-DCs.
Is it necessary fix my cells before permeabilization?
From my own experience I would say yes. The needed permabilization process opens up the membrane to let the detection antibody in, which means intracellular proteins can go out as well that it is very harsh on the cell and will kill them during the permabilization process. I have no experience with the proteins you mentioned but did staining for transcription factors and protein contained in granular vesicles. I hope this will help, if I stumble on a reference to confirm this i will let you know.
generally in my experiments after lineage staining , i fix the cells and proceed for permeabilization. fixed cells stained with lineage markers might show irregularities for certain antigens . Better study some of the flow cytometry brochures from standard companies to have an idea on this.
Here is good link to intracellular staining procedures:
https://www.thermofisher.com/us/en/home/references/protocols/cell-and-tissue-analysis/protocols/staining-intracellular-antigens-flow-cytometry.html
Different staining procedures can be done for nuclear and cytoplasmic proteins.
From my own experience for cytoplasmic staining 4% buffered PFA (Molecular grade-w/o methanol) 15 min at RT fixation, followed by permeabilization with 0.1% Triton X100 in PBS 15 min at RT works fine for monocytic cell lines. For nuclear staining eBioscience FOXP3 PFA contaning Fix/perm buffer 30 min on ice fixation, followed by 1x Perm (eBioscience hu FoxP3 kit) saponin-containing buffer 30 min at RT works fine for human nuclear proteins. For mice cells/ nuclear proteins detection I fix in FoxP3 Fix/perm buffer overnight at 4C. Noticed some problems with short-term fixation of mice primary prostate cells.
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