How does PI staining work? - (Jul/23/2007 )

Hi,
I don't understand how PI staining works. From what I know, PI intercalates with the DNA to give a red fluorescent stain, and this is detected in the flow cytometer when the cell passes through it.
What puzzles me is how the flow cytometer can distinguish between cells in sub G1 and those in G1 since they both contain the same amount of DNA (2n). I read somewhere that apoptotic cells have reduced membrane permeability to dyes; does this have anything to do with it?
I've tried searching pubmed books but i don't get any hits, so if anybody knows how PI works please reply! Really appreciate it.
Thanks!

PI staining for apoptotic/dead cell works in the same way as trypan blue exclusion. Viable cells have impermeable membrane to PI and it cannot bind DNA within the cell.

You seem to be talking about measuring DNA content following permeabilising the cell with cold ethanol/methanol. Sub-G1 cells are apoptotic so the DNA has been fragmented/degraded so there's less DNA than in viable cells.

Ceri

Ooh I see... Yes the protocol I was given requires fixing the cells with ethanol. Thanks so much for your help!

sorry to trouble u but i have one more question... for the cells shown to be in G1 phase, are they actually about to apoptosize as well? Since a viable cell won't be able to absorb the dye....