Why do you need to stain for both: DAPI and PI?  They both bind DNA and PI also binds RNA. There will be some crosstalk between dyes, even if you use proper filters.

PFA fixation  does not affect  too much PI or DAPI staining.

Hi Natallia,

I just wanted to have a way of representing my total cell count using DAPI. My PI positive cells will be the ones undergoing necroptosis. So I would plot % cell death (PI positive/DAPI positive). I suppose I could try to image in brightfield to get a total cell count if there will be cross talk. 

You will need to  use low PI concentrations and wash your cells really  really well with high serum buffer after PI  or DAPI staining as it will leak from dead cells after PFA fixation and stain fixed "live" before fixation cells.  Myself I would never do  that with PFA or other fixation. You might try to use fixable viability (amines binding- protein and DNA , etc  covalent binding) dyes instead (like Zombie Nir (far red))  that can separate dead cells before fixation  from cells that  died  when you add fixative.

Hi,