Maryalice, Our lab studies drug effects in bone marrow and this method is not used to evaluate viability but rather cell cycle. We use the paraformaldehyde to fix the shape of rat bone marrow cells, then permeabilize the cells to allow PI to penetrate for cell cycle evaluation. For bone marrow, much subpopulation analysis is done by forward and side scatter (since there is a relative paucity of specific anti-rat antibodies). Most methods that simply permeabilize the cells strip the a good portion of the cytoplasm off the cell and some methods leave bare nuclei. While this is good enough for cell cycle evaluation of pure cell populations, stripping the cytoplasm prevents identification of the affected subpopulation of bone marrow (eg erythroid toxicity vs myeloid toxicty) according to forward and side scatter. So in order to evaluate the effects of drugs on cell cycle of different subpopulations, we first experimented with a lot of different fixation/permeablization methods before we found one that gave a histogram similar to unfixed cells plus 100% PI uptake. During the evaluation process we discovered that paraformaldehyde fixation alone permeabilizes less than 25% of the cells. Most of these cells were not permeable prior to paraformadehyde fixation - therefore the paraformaldehyde fixation process also permeabilized some fraction of the cells in this complex mixture. Paraformaldehyde fixation can not be used prior to PI in rat bone marrow if the method is being used for viability, despite the fact that others have successfully used this method for some cell lines. The point I was trying to make is that each cell system needs to be evaluated independently to determine whether the method gives accurate results for the specific lab with the specific cell line and the information can not be extrapolated from one cell system to another without actual experimentation... Carol W. Johnson, DVM PhD Maryalice Stetler-Stevenson <stetler@box-s.nih.gov> on 04/23/2001 05:39:16 PM To: cyto-inbox cc: (bcc: Carol W Johnson/USKZO/PNU) Subject: Re: Paraformaldehyde and PI It still does not make sense to fix and then us PI for viability. >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 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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