A method for objectively quantifying propidium iodide exclusion in organotypic hippocampal slice cultures
| Autor: | R. Andrew Tasker, Denise F. Happ |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Male Programmed cell death N-Methylaspartate Cell Survival Excitotoxicity Hippocampus Cell Count Hippocampal formation Biology medicine.disease_cause Rats Sprague-Dawley Tissue Culture Techniques 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine medicine Image Processing Computer-Assisted Animals Viability assay Propidium iodide Neurons Cell Death Dose-Response Relationship Drug Staining and Labeling General Neuroscience Dentate gyrus 030104 developmental biology nervous system chemistry Microscopy Fluorescence NMDA receptor Female Neuroscience 030217 neurology & neurosurgery Densitometry Propidium |
| Zdroj: | Happ, D F & Tasker, A 2016, ' A method for objectively quantifying propidium iodide exclusion in organotypic hippocampal slice cultures ', Journal of Neuroscience Methods, vol. 269, pp. 1-5 . https://doi.org/10.1016/j.jneumeth.2016.05.006 |
| ISSN: | 1872-678X |
| Popis: | BACKGROUND: Organotypic hippocampal slice cultures (OHSCs) are an attractive in vitro model to examine mechanisms of neuronal injury, because the normal hippocampal architecture, function and cellular diversity are mostly preserved. The effects of exposure to excitotoxins such as N-methyl-d-aspartate (NMDA) on cell viability can be determined by propidium iodide (PI) staining.NEW METHOD: We describe a simple method to objectively quantify cell death in NMDA exposed slice cultures using PI that provides a standardized means of quantifying cell death in hippocampal subfields without the need to induce maximal cell death in each slice. The method employs separation of subfields using simple landmarks and densitometric quantification of PI intensity in 10 template-oriented counting fields.RESULTS: We show that exposure to increasing concentrations of NMDA results in a dose-dependent increase in PI uptake. Additionally, our method facilitates the comparison of cell death in different hippocampal subfields, such as dentate gyrus, CA1 and CA3. Our results show marked differences of PI uptake in the hippocampal regions with the CA1 area being most sensitive to NMDA-induced injury.COMPARISON WITH EXISTING METHOD(S): The method provides a standardized format for quantifying PI exclusion in OHSCs that can be applied to cultures of differing shapes and sizes, permits comparisons between hippocampal subfields and does not require induction of maximal cell death.CONCLUSION: The method of quantifying PI uptake described herein allows for an objective, quantitative and reproducible analysis and comparison of cell death in distinct regions of OHSCs. |
| Databáze: | OpenAIRE |
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