An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Abstract

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and subsequently digests this material, commonly through lysosomal degradation. Microglia are the resident immune cells of the central nervous system (CNS) whose phagocytic function has been described in a broad range of conditions from neurodegenerative disease (e.g., beta-amyloid clearance in Alzheimer's disease) to development of the healthy brain (e.g., synaptic pruning) [1][2][3][4][5][6] . The following protocol is an engulfment assay developed to visualize and quantify microglia-mediated engulfment of presynaptic inputs in the developing mouse retinogeniculate system 7 . While this assay was used to assess microglia function in this particular context, a similar approach may be used to assess other phagocytes throughout the brain (e.g., astrocytes) and the rest of the body (e.g., peripheral macrophages) as well as other contexts in which synaptic remodeling occurs (e.g. ,brain injury/disease). Video LinkThe video component of this article can be found at

Inhibition of Stat3‐mediated astrogliosis ameliorates pathology in an Alzheimer's disease model

“…To quantify the volume of engulfed Ab, fluorescence outside of astrocytes or microglia was subtracted from the image, and the remaining engulfed fluorescence was surface-rendered and calculated (Schafer et al, 2012(Schafer et al, , 2014. 3D volume surface renderings of each z-stack were created using Imaris 8.4.1 (Bitplane), and the volume of glial cells and Ab compartments (IC16 and methoxy-XO4) was determined from surface-rendered images.…”

“…3D volume surface renderings of each z-stack were created using Imaris 8.4.1 (Bitplane), and the volume of glial cells and Ab compartments (IC16 and methoxy-XO4) was determined from surface-rendered images. To quantify the volume of engulfed Ab, fluorescence outside of astrocytes or microglia was subtracted from the image, and the remaining engulfed fluorescence was surface-rendered and calculated (Schafer et al, 2012(Schafer et al, , 2014.…”

Sensory lesioning induces microglial synapse elimination via ADAM10 and fractalkine signaling

“…1g-o and Supplementary Fig. 3a) 1,31 . These inputs were largely localized within the microglia, but not yet significantly associated with lysosomes ( Fig.…”

Microglia Function in Central Nervous System Development and Plasticity

“…Using this system, the investigators observed microglial processes closely apposed to presynaptic inputs undergoing remodeling within the dLGN, data consistent with previous live imaging and EM data (Tremblay et al 2010). Subsequently, a novel in vivo assay was developed in mice to determine if microglia were engulfing inputs (Schafer et al 2012(Schafer et al , 2014. In this engulfment assay, presynaptic inputs within the dLGN are fluorescently labeled by intraocular injection of anterograde dyes that are highly stable and resistant to lysosomal degradation (i.e., cholera toxin b subunit conjugated to Alexa dyes).…”