Isolation of Mouse Primary Microglia by Magnetic-Activated Cell Sorting in Animal Models of Demyelination

Podsumowanie

Here, we present a protocol to isolate and purify primary microglia in animal models of demyelinating diseases, utilizing columnar magnetic-activated cell sorting.

Streszczenie

Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). Microglia can be divided into resting state and activated state and can rapidly change state in response to the microenvironment of the brain. Microglia will be activated under different pathological conditions and exhibit different phenotypes. In addition, there are many different subgroups of activated microglia and great heterogeneity between different subgroups. The heterogeneity mainly depends on the molecular specificity of microglia. Studies have revealed that microglia will be activated and play an important role in the pathological process of inflammatory demyelination. To better understand the characteristics of microglia in inflammatory demyelinating diseases such as multiple sclerosis and neuromyelitis optica spectrum disorder, we propose a perilesional primary microglial sorting protocol. This protocol utilizes columnar magnetic-activated cell sorting (MACS) to obtain highly purified primary microglia and preserve the molecular characteristics of microglia to investigate the potential effects of microglia in inflammatory demyelinating diseases.

Wprowadzenie

Microglia originate from yolk-sac progenitors, which reach the embryonic brain very early and participate in the development of the CNS^1,2. For instance, they are involved in synaptic pruning³ and regulating axonal growth⁴. They secrete factors that promote neuronal survival and help neuronal localization⁵. At the same time, they are involved in removing abnormal cells and apoptotic cells to ensure normal brain development⁶. In addition, as the immune-competent cells of the brain, microglia con....

Protokół

All the animal procedures have been approved by the Institute of Animal Care Committee of Tongji Medical College, Huazhong University of Science and Technology, China.

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, pipe....

Wyniki

Microglia isolated using CD11b beads have high purity
Microglial cells around the lesions in demyelination mouse models were isolated using the above-mentioned protocol and tested by flow cytometry. Cells are fluorescently labeled with CD11b-fluorescein isothiocyanate (FITC) and CD45-allophycocyanin (APC) to determine microglia in flow cytometry according to the manufacturer's instructions. There are multiple literatures demonstrating that CD11b and CD45 antibodies are enough to check for the p.......

Dyskusje

The protocol proposes a method to isolate microglia around the demyelinating lesions, which can help study the functional characteristics of microglia in inflammatory demyelinating diseases. Microglia captured using CD11b beads exhibit high purity and viability. Critical steps in the protocol include the precise localization of foci and optimal microglial purification. In protocol step 2.1, it is necessary to inject the NR solution 2 h before sacrificing the mouse to ensure that the lesions can be accurately displayed

Materiały

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
FlowJo	BD Biosciences	V10
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
NovoCyte Flow Cytometer	Agilent		A system consisting of various parts
NovoExpress	Agilent	1.4.1
PBS	BOSTER	PYG0021
Pentobarbital	Sigma-Aldrich	P-010
Stereomicroscope	MshOt	MZ62