I need to detect and measure the abundance of cytoplasmatic proteins (AIM2, GBP1, GBP4) and some nucleic proteins (IFI16)using direct detection by flow cytometry with fluorochrome-conjugated antibodies in human mo-DCs..
What permeabilizing agent is recommended in my case: saponin, digitonin, Triton, Tween 20 or another one?
Someone have some advices and experiences?
Hi Amanda, Yes first I would buy a commercially made paraformaldehyde solution as this chemical is highly carcinogenic when in powder form. Also heating it up takes a long time at 56C and the fumes are highly toxic, you could adjust the pH with sodium hyroxide which dissolves this very rapidly and then re-Ph back to 7, but the resulting solution is highly unstable even at -80C. There are numerous paraformaldehyde solutions available from flow cytometry companies available. I use Triton X-100 at 0.025% for intracellular antigen work but using 0.1% saponin will open the pores made in the cell more when staining with the antibodies, also adding saponin to the wash buffer helps reduce back background
Regards
Gary
I usually use the commercial Triton X-100 as a permeabilizer, and the results are not bad.
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