Quantification of Microglial Engulfment of Synaptic Material Using Flow Cytometry

J Vis Exp. 2024 May 31:(207). doi: 10.3791/66639.

Abstract

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this process; however, currently available methods for identifying microglial engulfment of synapses, such as immunohistochemistry (IHC) and imaging, are laborious and time-intensive. To address this challenge, herein we present in vitro and in vivo* assays that allow fast and high-throughput quantification of microglial engulfment of synapses using flow cytometry. In the in vivo* approach, we performed intracellular vGLUT1 staining following fresh cell isolation from adult mouse brains to quantify engulfment of vGLUT1+ synapses by microglia. In the in vitro synaptosome engulfment assay, we used freshly isolated cells from the adult mouse brain to quantify the engulfment of pHrodo Red-labeled synaptosomes by microglia. These protocols together provide a time-efficient approach to quantifying microglial engulfment of synapses and represent promising alternatives to labor-intensive image analysis-based methods. By streamlining the analysis, these assays can contribute to a better understanding of the role of microglia in synaptic refinement in different disease models.

Publication types

  • Video-Audio Media

MeSH terms

  • Animals
  • Brain / cytology
  • Flow Cytometry* / methods
  • Mice
  • Microglia* / cytology
  • Microglia* / metabolism
  • Synapses* / chemistry
  • Synapses* / physiology
  • Synaptosomes / metabolism
  • Vesicular Glutamate Transport Protein 1 / analysis
  • Vesicular Glutamate Transport Protein 1 / metabolism

Substances

  • Vesicular Glutamate Transport Protein 1
  • Slc17a7 protein, mouse