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

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 CX₃CR₁^creERT2 × iDTR or CX₃CR₁^creERT2 × iDTR × tdTomato^flox 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.

Graphical abstract

Figure 2

Hamilton syringe attached and held in place to the syringe pump

Figure 3

Attachment of polyethylene tubing to the Hamilton syringe needle

Figure 4

Tubing with needle end connected to Hamilton syringe held in place to stereotaxic rig

Figure 5

Tubing filled with working solution to be injected using stereotaxic setup

Figure 1

Setup of stereotaxic rig used for brain injections in mice

Figure 6

Schematic overview of hippocampus dissection

Figure 7

Papain dissociation buffer added to diced hippocampi in Petri dish

Figure 8

Representative FACS gating and tdTomato-positive microglia sorting strategy from an uninjured tamoxifen-treated CX₃CR₁^creERT2 × iDTR × tdTomato^flox mouse with resident microglia

Figure 9

Depletion of microglia in CX₃CR₁^creERT2 × iDTR × tdTomato^flox mice (A) Experimental timeline for tamoxifen treatment followed by saline vehicle or DT administration via IP injection for microglia depletion in CX₃CR₁^creERT2 × iDTR × tdTomato^flox mice. (B) tdTomato^pos microglia numbers in the dentate gyrus of saline (vehicle) versus DT-treated CX₃CR₁^creERT2 × iDTR × tdTomato^flox 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 tdTomato^pos microglia in CX₃CR₁^creERT2 × iDTR × tdTomato^flox mice. Scale bar, 400 μm.