Rapid and Efficient Enrichment of Mouse Spinal Cord Microglia

Summary

Microglia are regarded as some of the most versatile cells in the body, capable of morphological and functional adaptation. Their heterogeneity and multifunctionality enable the maintenance of brain homeostasis, while also being linked to various neurological pathologies. Here, a technique for purifying spinal cord microglia is described.

Abstract

The vertebral column defines a vertebrate and shapes the spinal canal, a cavity that encloses and safeguards the spinal cord. Proper development and function of the mammalian central nervous system rely significantly on the activity of resident macrophages known as microglia. Microglia display heterogeneity and multifunctionality, enabling distinct gene expression and behavior within the spinal cord and brain. Numerous studies have explored cerebral microglia function, detailing purification methods extensively. However, the purification of microglia from the spinal cord in mice lacks a comprehensive description. In contrast, the utilization of a highly purified collagenase, as opposed to an unrefined extract, lacks reporting within central nervous system tissues. In this study, the vertebral column and spinal cord were excised from 8-10 week-old C57BL/6 mice. Subsequent digestion employed a highly purified collagenase, and microglia purification utilized a density gradient. Cells underwent staining for flow cytometry, assessing viability and purity through CD11b and CD45 staining. Results yielded an average viability of 80% and a mean purity of 95%. In conclusion, manipulation of mouse microglia involved digestion with a highly purified collagenase, followed by a density gradient. This approach effectively produced substantial spinal cord microglia populations.

Introduction

The defining characteristic of vertebrates is the vertebral column or spine, in which the notochord has been replaced by a sequence of segmented bones called vertebrae, divided by intervertebral discs. This succession of osseous material shapes the spinal canal, a cavity that encloses and protects the spinal cord¹. In the genus Rodentia, the spine is usually formed by seven cervical vertebrae, thirteen thoracic vertebrae, six lumbar vertebrae, and a variable number of caudal vertebrae^2,3. The length of the spinal cord is similar to that of the spine, and the terminal filum is a....

Protocol

The study was conducted in accordance with the official Mexican standard NOM-062-ZOO-1999 and the guide for the care and use of laboratory animals. Approval for the study was obtained from the Research, Ethics, and Biosafety Committees of the Mexico Children's Hospital (HIM/2023/006) and the Research and Bioethics Committee of the General Hospital of Mexico Eduardo Liceaga (DI/21/501/04/62). Three C57BL/6 mice aged 6 to 8 weeks were obtained from the Mexico Children's Hospital, where they were raised under isolated conditions in ventilated racks, in compliance with institutional animal care and use guidelines.

1. Preparation o....

Results

Utilizing mouse spinal cord tissue, enzymatic digestion was performed using a mixture highly enriched with collagenase and thermolysin. The resulting digested tissue underwent passage through a 40 µm filter to eliminate undigested material. The collected cells were enriched through a Percoll density gradient, with 90% in the lower portion and 45% in the upper portion. The microglia-enriched cells within the interface were then stained with CD45 and CD11b antibodies and subjected to flow cytometric analysis (

Discussion

Numerous protocols have been developed for the study of microglia due to their significance in brain homeostasis. In these methods, microglia are typically sourced from the cerebral hemispheres of embryonic or neonatal rats and mice¹⁷. A limited number of studies have addressed the purification of microglia from the spinal cords of adult mice^13,14. These techniques involve enzymatic digestion using collagenase and/or papain along with DNAs.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15 mL collection tubes	Corning, USA	430790
2 mL microtubes	Axygen, USA	MCT-200-G
2.4G2 anti-FcR	BioLegend, USA	101302
50 mL collection tubes	Corning, USA	430829
70% ethanol
Antibiotic-Antimycotic (penicillin, streptomycin, amphotericin b)	Gibco, USA	15240062
Antibody CD11b eFluor 450 anti-mouse	eBioscience, USA	48-0112
Antibody CD45 PerCP anti-mouse	BioLegend, USA	103130
Balanced salt solution (PBS) calcium- magnesium-free	Corning, USA	46-013-CM
Blue Cell Strainer 40 μm	Corning, USA	352340
Costar 6-well Clear Not Treated	Corning, USA	CLS3736
Coverslips
Digital Heating Shaking Drybath	Thermo Scientific Digital HS Drybath, USA	88870001
Dissecting forceps for microsurgery	FT by DUMONT
DNase	Roche, USA	4536282001
Dulbecco´s Modified Eagle´s Medium-high glucose (DMEM)	Merck, USA	D6429
Electric shaver
FACS tube	Thermo, USA	352058
Fetal bovine serum (FBS)	PAN Biotech, Alemania	P30-3306
Flow cytometer Cytoflex	Beckman Coulter
Hank’s balanced salt solution	Merck, USA	H2387
L-glutamine	Corning, USA	15393631
Liberase TM	Roche, USA	5401119001
Neubauer chamber Counting Chambers	China	1103
Pentobarbital
Percoll	Merck, USA	17089101	density gradient centrifugation
Poly-L-lysine solution	Merck, USA	P8920
Scalpel No. 25	HERGOM, Mexico	H23
Snaplock Microcentrifuge Tubes 2 mL	Axygen, USA	10011-680
Stereoscopic microscope	Velab, Mexico	HG927831
Straight surgical scissors (10 cm)	HERGOM, Mexico
Straight Vannas scissors	HERGOM, Mexico
Triton X100	Merck, USA	X100
Trypan blue Stain 0.4%	Merck, USA	15250-061
Vortex mixer	DLAB, China	8031102000
Zombie Aqua Fixable Viability Kit	BioLegend, USA	423102	amine-reactive fluorescent dye staining