Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures

Summary

Primary microglia cultures are commonly used to evaluate new anti-inflammatory molecules. The present protocol describes a reproducible and relevant method to magnetically isolate microglia from neonate pups.

Abstract

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. Microglia can adopt a large spectrum of activation phenotypes. Moreover, microglia that endorse pro-inflammatory phenotype is associated with both neurodevelopmental and neurodegenerative disorders. In vitro studies are widely used in research to evaluate potential therapeutic strategies in specific cell types. In this context studying microglial activation and neuroinflammation in vitro using primary microglial cultures is more relevant than microglial cell lines or stem-cell-derived microglia. However, the use of some primary cultures might suffer from a lack of reproducibility. This protocol proposes a reproducible and relevant method of magnetically isolating microglia from neonate pups. Microglial activation using several stimuli after 4 h and 24 h by mRNA expression quantification and a Cy3-bead phagocytic assay is demonstrated here. The current work is expected to provide an easily reproducible technique for isolating physiologically relevant microglia from juvenile developmental stages.

Introduction

Microglia are the central nervous system resident macrophage-like cells derived from erythropoietic precursors of the yolk sac that migrate to the neuroepithelium during early embryonic development¹. Apart from their immunity functions, they also play a significant role during neurodevelopment, particularly for synaptogenesis, neuronal homeostasis, and myelination². In adulthood, microglia develop long cellular processes to scan the environment continuously. In case of homeostasis ruptures such as brain injury or brain disease, microglia can change their morphological appearance to adopt an amoeboid shape, migrate to the....

Protocol

The protocol was approved, and all the animals were handled according to the institutional guidelines of Institut National de la Santé et de la Recherche Scientifique (Inserm, France). Magnetic isolation of microglia from the brains of 24 OF1 mouse pups (both male and female) at P8, divided into 6-well, 12-well, or 96-well plates, are presented. The experimental work was performed under a hood to maintain sterile conditions.

1. Preparation of sterile solutions for isolation and cel.......

Discussion

The current work presents a primary microglial cell culture using magnetically sorted CD11b+ cells. In addition to the microglial functional evaluation (RT-qPCR and phagocytic assays), microglial culture purity was also determined.

Classical microglia cell cultures are commonly generated from P1 or P2 rodent neonate brain and co-culture with astrocytes for at least 10 days. Microglia are then separated mechanically using an orbital shaker. The method to isolate and culture microglia in vit.......

Materials

Name	Company	Catalog Number	Comments
Anti mouse ACSA-2 PE Vio 615	Miltenyi Biotec	130-116-246
Anti mouse CD11b BV421	Sony Biotechnology	1106255
Anti mouse CD45 BV510	Sony Biotechnology	1115690
Anti mouse CX3CR1 PE Cy7	Sony Biotechnology	1345075
Anti mouse NeuN PE	Milli-Mark	FCMAB317PE
anti mouse O4 Vio Bright B515	Miltenyi Biotec	130-120-016
BD Cytofix/Cytoperm permeabilization kit	BD Biosciences	554655
Bovine Serum Albumin	Miltenyi Biotec	130-091-376
CD11b (Microglia) MicroBeads, h, m	Miltenyi Biotec	130-093-634
Confocal microscope	Leica TCS SP8
D-PBS (10x)	Thermo Scientific	14200067
EDTA	Sigma-Aldrich	E1644
Falcon Cell culture 12-well plate, flat bottom + lid	Dutscher	353043
Falcon Cell culture 96-well plate, flat bottom + lid	Dutscher	353072
Falcon tubes 50 mL	Dutscher	352098
Fc blocking reagent (Mouse CD16/32)	BD Biosciences	553142
Fluorescence microscope	Nikon ECLIPSE TE300
gentleMACS C Tubes (4 x 25 tubes)	Miltenyi Biotec	130-096-334
gentleMACS Octo Dissociator with Heaters	Miltenyi Biotec	130-096-427
Hanks' Balanced Salt Solution (HBSS) +CaCl2 +MgCl2 10x	Thermo Scientific	14065049
Hanks' Balanced Salt Solution (HBSS) -CaCl2 -MgCl2 10x	Thermo Scientific	14185045
iQ SYBR Green Supermix	Bio-rad	1725006CUST
Iscript c-DNA synthesis	Bio-rad	1708890
Latex beads, amine-modified polystyrene, fluorescent red	Sigma-Aldrich	L2776-1mL
Lipopolysaccharides (LPS) from Escherichia coli O55:B5	Sigma-Aldrich	L2880
Macrophage-SFM serum-free medium	Thermo Scientific	12065074
MACS BSA Stock Solution	Miltenyi Biotec	130-091-376
MACS SmartStrainers (70 μm), 4 x 25 pcs	Miltenyi Biotec	130-110-916
Mouse IgG1 PE	Millipore	MABC002H
Mouse IgG2a PE Cy7	Sony Biotechnology	2601265
Mouse IL1 beta	Miltenyi Biotec	130-101-684
Multi-24 Column Blocks	Miltenyi Biotec	130-095-691
MultiMACS Cell24 Separator	Miltenyi Biotec
Neural Tissue Dissociation Kit - Papain	Miltenyi Biotec	130-092-628
Nucleocounter NC-200	Chemometec
Nucleospin RNA Plus XS	Macherey Nagel	740990.5
Nun EZFlip Top Conical Centrifuge Tubes	Thermo Scientific	362694
OPTILUX Petri dish - 100 x 20 mm	Dutscher	353003
Pénicilline-streptomycine (10 000 U/mL)	Thermo Scientific	15140122
Rat IgG2b, k BV421	BD Biosciences	562603
Rat IgG2b, k BV510	Sony Biotechnology	2603230
REA control (S) PE vio 615	Miltenyi Biotec	130-104-616
REA control (S) Vio Bright B515	Miltenyi Biotec	130-113-445
Recombinant Mouse IFN-gamma Protein	R&D System	485-MI
Recombinant Mouse IL-10 Protein	R&D System	417-ML
Recombinant Mouse IL-4 Protein	R&D System	404-ML
RIPA Buffer	Sigma-Aldrich	R0278
Viability probe (FVS780)	BD Biosciences	565388