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Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death
Published on: April 24, 2011
A method for characterising cell death in vitro by combining propidium iodide staining with immunohistochemistry
Corinne Brana1, Chris Benham, Lars Sundstrom
1Laboratoire 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
Insights
Researchers developed a method to preserve propidium iodide (PI) signals in fixed tissues, enabling cell death characterization. This technique allows detailed analysis of dying cells and their phenotypes in various experimental models.
Area of Science:
- Neuroscience
- Cell Biology
- Histology
Background:
- Propidium iodide (PI) is a vital dye used to identify cells lacking an intact plasma membrane.
- Preserving PI signal in fixed tissues for subsequent analysis has been a challenge.
Purpose of the Study:
- To develop and validate a method for preserving PI fluorescence in fixed tissues.
- To enable co-localization of PI-positive cells with specific cellular markers.
Main Methods:
- Organotypic hippocampal slice cultures were subjected to oxygen and glucose deprivation.
- Paraformaldehyde fixation was used to preserve PI signal.
- Immunohistochemistry was performed to detect calpain-I mediated spectrin breakdown products (BDPs).
- Confocal laser microscopy was employed for single-cell analysis.
Main Results:
- The developed method successfully preserved PI signal in paraformaldehyde-fixed tissue.
- PI-positive cells were correlated with BDP expression at the single-cell level.
- The technique allowed for the phenotypic characterization of PI-positive cells.
Conclusions:
- This method allows for the preservation and characterization of PI-labeled dying cells in fixed tissues.
- It facilitates the study of cell death mechanisms and phenotypes in various in vitro and in vivo models.
- The technique provides a snapshot of cell viability at different time points after insult.
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.
