Your experience will differ depending on the fixative you really use, osmotic pressure etc. Remember that paraformaldehyde is an insoluble white powder..... there are long discussions about this already out here. PI will detect membrane permeabilisation which can be temporarily (be careful when detaching cells). Thus cells do not need to be dead just because they take up PI and the stain has been used successfully to measure transient pore formation. Crosslinking the membrane does not permeabilize it necessarily. I have seen a lot of formaldehyde fixed cells that did not stain for PI. As perhaps an easier alternative you might want to incubate your cells with CFSE in culture and wash it off before detaching the cells. The cells alive at point of detachment or fixation will remain bright green. You can than combine this with PI if you want. Regards Gerhard -----Original Message----- From: Tomas Corcoran [SMTP:mascor@gofree.indigo.ie] Sent: Tuesday, April 17, 2001 7:44 PM To: Cytometry Mailing List Subject: Paraformaldehyde and PI 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. mascor@gofree.indigo.ie
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