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Protocol to assess engulfment and degradation of synaptosomes by murine microglia in vitro
Alessandro Matera1, Anne-Claire Compagnion1, Rosa Chiara Paolicelli1
1University of Lausanne, Department of Biomedical Sciences, 1005 Lausanne, Switzerland.
Abstract:
Microglia, the innate immune cells of the central nervous system, refine neuronal circuitries both during brain development and in neurodegenerative diseases. Here, we present a protocol to independently assess the engulfment and degradation of synaptosomes by murine microglia, both in fixed and live samples, using confocal imaging. We describe steps for isolating primary microglia, preparing and conjugating synaptosomes with pHrodo, and performing an uptake and degradation assay. We then detail procedures for analyzing synaptosome uptake and degradation using ImageJ software. For complete details on the use and execution of this protocol, please refer to Matera, Compagnion, et al.1.
Insights
This study details a new protocol for measuring how microglia, the brain's immune cells, engulf and break down synaptosomes. This method aids research into brain development and neurodegenerative diseases.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia are key immune cells in the central nervous system.
- Microglia play roles in brain development and neurodegenerative diseases.
- Understanding microglial function in synaptosome processing is crucial.
Purpose of the Study:
- To present a novel protocol for assessing microglial engulfment and degradation of synaptosomes.
- To enable independent measurement of these two processes in both fixed and live samples.
- To provide a method applicable to studies of brain development and neurodegeneration.
Main Methods:
- Isolation of primary murine microglia.
- Preparation and pHrodo conjugation of synaptosomes.
- Confocal imaging-based uptake and degradation assay.
- ImageJ software for data analysis.
Main Results:
- The protocol allows for independent quantification of synaptosome engulfment by microglia.
- The protocol enables assessment of synaptosome degradation within microglia.
- The method is validated for use with both fixed and live cellular samples.
Conclusions:
- This protocol offers a robust method for studying microglial phagocytic activity.
- The technique is valuable for investigating microglial roles in health and disease.
- The developed assay provides insights into neuronal-glial interactions.
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