title: A method for characterising cell death in vitro by combining propidium iodide staining with immunohistochemistry.
abstract: The fluorescent exclusion dye propidium iodide (PI) is widely used as a vital dye in tissue culture systems and labels the nucleus in dying cells which lack an intact plasma membrane. We have developed a method, which allows the preservation of the PI signal in paraformaldehyde-fixed tissue, enabling subsequent immunohistochemical characterisation of labelled cells. We have tested this method in a model of ischemia based on oxygen and glucose deprivation in organotypic hippocampal slice cultures, in combination with immunocytochemical detection of calpain-I mediated spectrin breakdown products (BDPs). Using confocal laser microscopy it was possible to correlate at the single cell level which cells were PI positive and which cells expressed BDPs. This method can also be used with other immunocytochemical markers to determine the phenotype of cells, which accumulate PI in vitro. By fixing tissue at different times after insults, it is possible to obtain a 'snapshot' of viability at any time during the experimental protocol and subsequently characterise those cells which had accumulated PI at the time of fixation. The technique may also prove useful in characterising cell death in other in vitro and in vivo systems.
authors.affiliation: Laboratoire d'Epileptologie Expérimentale et Clinique (EA2967), Université Victor Segalen Bordeaux 2, 146 Rue Léo Saignat, Boîte 78, 33076 Bordeaux, France. corinne.brana@ar-luni.u-bordeaux2.fr
authors.forename: Corinne
authors.initials: C
authors.lastname: Brana
authors.forename: Chris
authors.initials: C
authors.lastname: Benham
authors.forename: Lars
authors.initials: L
authors.lastname: Sundstrom
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D016923:Cell Death / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D020925:Hypoxia-Ischemia, Brain / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D007150:Immunohistochemistry / Q000295:instrumentation / Q000379:methods*; D008856:Microscopy, Fluorescence; D009410:Nerve Degeneration / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009924:Organ Culture Techniques / Q000295:instrumentation / Q000379:methods*; D011419:Propidium; D051381:Rats; D017208:Rats, Wistar
chemical_list: D011419:Propidium