Isolating Microglia from Mouse Brain Demyelinating Lesions Using Magnetic Activated Cell Sorting

Abstract

Source: Zhang, H. et al. Isolation of Mouse Primary Microglia by Magnetic-Activated Cell Sorting in Animal Models of Demyelination. J. Vis. Exp. (2022).

This video demonstrates a protocol for extracting microglial cells from demyelinating lesions in mouse brains using magnetic cell sorting. The protocol involves dissecting the lesioned brain tissue containing microglial cells, treating the tissue with specific enzymes to obtain a single-cell suspension, and utilizing microglial receptor-specific magnetic beads to isolate the microglial cells.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Materials

1. Prepare the following solutions before beginning the protocol:
   1. Prepare the loading buffer by adding fetal bovine serum (FBS, 2%) to phosphate-buffered saline (PBS).
   2. Add neutral red (NR) dye (final 1%) to PBS.
2. Prepare the following solutions using the commercially available Adult Brain Dissociation Kit (see the Table of Materials).
   1. To prepare enzyme mix 1, pipette 1.9 mL of Buffer Z and 50 μL of ....

Results

Figure 1: Images of the focal demyelinating lesions labeled by the neutral red dye.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL Micro Centrifuge Tubes	BIOFIL	CFT001015
15 mL Centrifuge Tubes	BIOFIL	CFT011150
50 mL Centrifuge Tubes	BIOFIL	CFT011500
70 µm Filter	Miltenyi Biotec	130-095-823
Adult Brain Dissociation Kit, mouse and rat	Miltenyi Biotec	130-107-677
C57BL/6J Mice	SJA Labs
CD11b (Microglia) Beads, human and mouse	Miltenyi Biotec	130-093-634
Fetal Bovine Serum	BOSTER	PYG0001
MACS MultiStand	Miltenyi Biotec	130-042-303
MiniMACS Separator	Miltenyi Biotec	130-042-102
MS columns	Miltenyi Biotec	130-042-201
Neutral Red	Sigma-Aldrich	1013690025
PBS	BOSTER	PYG0021
Pentobarbital	Sigma-Aldrich	P-010
Stereomicroscope	MshOt	MZ62