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A method for characterising cell death in vitro by combining propidium iodide staining with immunohistochemistry

A method for characterising cell death in vitro by combining propidium iodide staining with immunohistochemistry
A method for characterising cell death in vitro by combining propidium iodide staining with immunohistochemistry
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.
organotypic cultures, propidium iodide, cell viability, confocal microscopy, immunocytochemistry
1385-299X
109-114
Brana, Corinne
7e18f85a-949a-4cbf-8117-26d68ecfac1b
Benham, Chris
f184f393-d7a9-408c-a480-7c3ea63a3654
Sundstrom, Lars
6d63d054-c735-4fcd-9cfc-2d41d68a06eb
Brana, Corinne
7e18f85a-949a-4cbf-8117-26d68ecfac1b
Benham, Chris
f184f393-d7a9-408c-a480-7c3ea63a3654
Sundstrom, Lars
6d63d054-c735-4fcd-9cfc-2d41d68a06eb

Brana, Corinne, Benham, Chris and Sundstrom, Lars (2002) A method for characterising cell death in vitro by combining propidium iodide staining with immunohistochemistry. Brain Research Protocols, 10 (2), 109-114. (doi:10.1016/S1385-299X(02)00201-5).

Record type: Article

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.

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More information

Published date: 2002
Keywords: organotypic cultures, propidium iodide, cell viability, confocal microscopy, immunocytochemistry

Identifiers

Local EPrints ID: 27538
URI: http://eprints.soton.ac.uk/id/eprint/27538
ISSN: 1385-299X
PURE UUID: e826f30d-4eaf-455f-9322-763d81a5818c

Catalogue record

Date deposited: 25 Apr 2006
Last modified: 15 Jul 2019 19:12

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