Isolation of Hypothalamic Microglia from Freshly Perfused Adult Mouse Brain by Magnetic-Activated Cell Sorting

Summary

We describe a protocol for isolating microglial cells from the mouse hypothalamus (or equivalent small brain structures) using magnetically activated cell sorting (MACS), in a relatively short time. The MACS-sorted hypothalamic microglia can be used for ex vivo analysis and can be plated to perform in vitro assays.

Abstract

Microglia, as the resident macrophages of the brain, are essential for maintaining brain homeostasis. They shape neuronal circuits during development, survey their environment for debris or dead cells, as well as respond to infection and injury in the brain, among many other functions. However, their important role in neurodevelopment and synaptic plasticity and pathophysiology has not been fully defined, highlighting the need for further investigation. To gain a more comprehensive understanding of the role of microglia in these processes, we need to isolate microglia and characterize them genetically, metabolically, and functionally. However, the isolation of microglia from adult mice, especially from small brain structures, is challenging as they represent a small percentage of the total brain cells, and the yield of isolated microglia is often too low. Here, the magnetic isolation of microglia using CD11b⁺ microbeads allows us to sort microglial cells from the hypothalamus of a freshly perfused adult mouse brain. The current method allows us to achieve relatively high purity and yield in a short period while maintaining cell viability.

Introduction

Microglia correspond to 5-20% of the total neural cells and are the only glial cells that originate from erythromyeloid progenitors in the yolk sac and start to colonize the developing brain around embryonic day E9.5^1,2. They are long-lived cells with the ability to undergo self-renewal slowly, independently of bone-marrow-derived cells³. As some of the most highly dynamic cells, they are capable of acquiring diverse phenotypes in response to contextual and environmental cues^2,4. Among the signals able to modulate their activity....

Protocol

All animal experiments described were conducted in strict compliance with the European Union recommendations (2013/63/EU) and were approved by the local ethical committee of the University of Bordeaux (CEEA50) and the French Ministry of Higher Education, Research, and Innovation (non-technical summary of approved project NTS-FR-619193 v.1, 23-12-2022).

The following protocol is performed on adult C57BL/6 mice with an average age of 2-4 months. However, it could be performed in all mice ages an.......

Discussion

The current protocol presents the isolation of hypothalamic microglia from freshly perfused adult mouse brain by Magnetic-Activated Cell Sorting. The results presented above confirm the purity and viability of isolated cells, as well as the efficacy of this method to functionally characterize microglia ex vivo, e.g. through phagocytic activity and ROS production quantification. Classical methods for the isolation of a specific cell population could also be used for microglial cells. Density gradient fractioning .......

Materials

Name	Company	Catalog Number	Comments
Adult Brain Dissociation Kit	Miltenyi	130-107-677	The kit contains buffer Z, buffer Y, enzymes A and P, Debris Removal Solution, buffer A (to dissolve enzyme A).
Albumin Bovine FrV BSA	EuroMedex	04-100-812-C
CD11b (Microglia) MicroBeads, human and mouse - small size	Miltenyi	130-093-636
CellROX Green Flow Cytometry Assay Kit	Invitrogen	C10492
Centrifuge Tube, Snap-Pop Lid 15 mL	CellTreat	978449
DPBS, calcium, magnesium, glucose, pyruvate	Gibco	14287-072
gentleMACS C Tubes	Miltenyi	130-093-237
gentleMACS Octo Dissociator with Heaters	Miltenyi	130-096-427
Halt Phosphatase Inhibitor Cocktail (100x)	Thermofisher	78420
Halt Protease Inhibitor Cocktail, EDTA free (100x)	Thermofisher	78437
HBSS (10x), calcium, magnesium, no phenol red	Thermofisher	14065056
Latex beads, carboxylate-modified polystyrene, fluorescent red	Sigma-Aldrich	L3280
LightCycler 480 SYBR Green I Master	Roche	4707516001	This reagent was used to perform PCR.
MACS SmartStrainers (70 µm)	Miltenyi	130-110-916
Micro BCA Protein Assay Kit	Thermofisher	23235
M-PER Mammalian Extraction Buffer	Thermofisher	78503
MS Columns	Miltenyi	130-042-201	Referred as small columns in the protocol.
MultiMACS Cell24 Separator Plus	Miltenyi	130-098-637
PBSS, pH 7.4	Thermofisher	10010023
qScript XLT cDNA SuperMix	Quanta biosciences	733-1177	The kit was used to syntesize cDNA.
ReliaPrep RNA Miniprep Systems	Promega	Z6011	The kit contains 1-Thioglycerol and BL buffer (referred as lysis buffer in the protocol) and it was used to isolate total RNA.
Vacutainer safety-lok 21 G	Becton Dickinson	367282