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. 2020 Dec 13;1(3):100211.
doi: 10.1016/j.xpro.2020.100211. eCollection 2020 Dec 18.

Protocol for brain-wide or region-specific microglia depletion and repopulation in adult mice

Emily F Willis  1 , Jana Vukovic  1
Affiliations

Protocol for brain-wide or region-specific microglia depletion and repopulation in adult mice

Emily F Willis et al. STAR Protoc. .

Abstract

The advent of tools enabling the direct manipulation of microglia has furthered our understanding of their role in health and disease. Here, we present a detailed protocol allowing for microglia turnover in adult CX3CR1creERT2 × iDTR or CX3CR1creERT2 × iDTR × tdTomatoflox mice, either in a brain-wide or region-specific manner, and their subsequent isolation for downstream applications. This protocol may be used to explore microglia biology and their putative region-specific heterogeneous functional diversity, expanding our understanding of their importance in various neuroinflammatory conditions. For complete details on the use and execution of this protocol, please refer to Willis et al. (2020).

Keywords: Flow Cytometry/Mass Cytometry; Immunology; Model Organisms; Neuroscience.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 2
Figure 2
Hamilton syringe attached and held in place to the syringe pump
Figure 3
Figure 3
Attachment of polyethylene tubing to the Hamilton syringe needle
Figure 4
Figure 4
Tubing with needle end connected to Hamilton syringe held in place to stereotaxic rig
Figure 5
Figure 5
Tubing filled with working solution to be injected using stereotaxic setup
Figure 1
Figure 1
Setup of stereotaxic rig used for brain injections in mice
Figure 6
Figure 6
Schematic overview of hippocampus dissection
Figure 7
Figure 7
Papain dissociation buffer added to diced hippocampi in Petri dish
Figure 8
Figure 8
Representative FACS gating and tdTomato-positive microglia sorting strategy from an uninjured tamoxifen-treated CX3CR1creERT2 × iDTR × tdTomatoflox mouse with resident microglia
Figure 9
Figure 9
Depletion of microglia in CX3CR1creERT2 × iDTR × tdTomatoflox mice (A) Experimental timeline for tamoxifen treatment followed by saline vehicle or DT administration via IP injection for microglia depletion in CX3CR1creERT2 × iDTR × tdTomatoflox mice. (B) tdTomatopos microglia numbers in the dentate gyrus of saline (vehicle) versus DT-treated CX3CR1creERT2 × iDTR × tdTomatoflox mice (t = 17.10, df = 8, p < 0.0001, ~90% depletion; unpaired Student’s t test). Data are represented as means ± SEM. (C) Representative confocal images of tdTomatopos microglia in CX3CR1creERT2 × iDTR × tdTomatoflox mice. Scale bar, 400 μm.
See this image and copyright information in PMC

References

    1. Parkhurst C.N., Yang G., Ninan I., Savas J.N., Yates J.R., 3rd, Lafaille J.J., Hempstead B.L., Littman D.R., Gan W.B. Microglia promote learning-dependent synapse formation through brain-derived neurotrophic factor. Cell. 2013;155:1596–1609. - PMC - PubMed
    1. Willis E.F., MacDonald K.P.A., Nguyen Q.H., Labrador Garrido A., Gillespie E.R., Harley S.B.R., Bartlett P.F., Schroder W., Yates A.G., Anthony D.C. Repopulating microglia promote brain repair in an IL-6-dependent manner. Cell. 2020;180:833–846.e16. - PubMed

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