Contrary to a variety of posts to this list, while paraformaldehyde does indeed
kill cells, it does not necessarily permeabilize them enough to allow PI into
all of the cells. For rat bone marrow cells, less than 25% of the cells take up
PI following 0.5% paraformaldehyde fixation (as evaluated under a fluorescent
microscope), even though 100% of the cells are indeed dead. Additional
permeabilization is needed to make all the cells pick up the dye.
There may be some variability between cell lines, etc. It is ALWAYS best to
actually look at the cells when a new method is tried.
Carol W. Johnson, DVM PhD
Pharmacia
Kalamazoo, MI
Maryalice Stetler-Stevenson <stetler@box-s.nih.gov> on 04/18/2001 03:31:37 PM
To: cyto-inbox
cc: (bcc: Carol W Johnson/USKZO/PNU)
Subject: Re: Paraformaldehyde and PI
PI is only excluded from live cells that are able to maintain their
cell membranes. Paraformaldehyde does kill cells.Therefore, fixing
live cells kills them. Then PI accurately demonstrates they are dead.
Who ever told you otherwise knows absolutely nothing about PI
exclusion from cells.
Maryalice
>I wonder whether anyone has experience or opinions on this :
>
>Does 0.5-2 %paraformaldehyde fixation of endothelial cells or tumour
>cells permeabilise cells to PI or not.Different sources say yes and
>no , but we have found that after removal of adherent cells from
>culture plates with 2mM EDTA and fixation that almost all cells
>stain with PI . Does paraformaldehyde fixation preclude meaningful
>viability assessment or not .
>
>Many thanks.
>
><mailto:mascor@gofree.indigo.ie>mascor@gofree.indigo.ie
>
Maryalice Stetler-Stevenson
Director Flow Cytometry Unit
Laboratory of Pathology, NCI, NIH
"Learn the rules so you know how to break them properly."
The Dalai Lama
PI is only excluded from live cells that are able to maintain their cell
membranes. Paraformaldehyde does kill cells.Therefore, fixing live cells kills
them. Then PI accurately demonstrates they are dead. Who ever told you otherwise
knows absolutely nothing about PI exclusion from cells.
Maryalice
I wonder whether anyone has experience or opinions on this :
Does 0.5-2 %paraformaldehyde fixation of endothelial cells or tumour cells
permeabilise cells to PI or not.Different sources say yes and no , but we
have found that after removal of adherent cells from culture plates with 2mM
EDTA and fixation that almost all cells stain with PI . Does paraformaldehyde
fixation preclude meaningful viability assessment or not .
Many thanks.
<mailto:mascor@gofree.indigo.ie>mascor@gofree.indigo.ie
Maryalice Stetler-Stevenson
Director Flow Cytometry Unit
Laboratory of Pathology, NCI, NIH
"Learn the rules so you know how to break them properly."
The Dalai Lama
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