TITLE: Isolation and Phenotyping of Adult Mouse Microglial Cells
AUTHORS: Kathleen Grabert, Barry W. McColl

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Microglia are the resident macrophages of the central nervous system parenchyma and fulfill crucial roles in brain development, homeostasis, and inflammation. The isolation of a pure microglia population from brain tissue enables the examination of microglial phenotypes without the interference of other cell populations. Microglial extractions from the neonatal brain have been described in various protocols, yet the more established and complex adult mouse brainposes a greater challenge. Here we describe a refined protocol including enzymatic and mechanical dissociation of adult mouse brain tissue and removal of myelin by Percoll density gradient. Microglial cells were subsequently extracted by an immunomagnetic approach. This isolation procedure enables the use of functionally viable cells for various applications such as cell culture, flow cytometry, functional assays including bacteria- or bead-based phagocytosis, stimulation assays, and transcriptome profiling techniques such as qRT-PCR and microarray/RNA sequencing.</div></div>

[STEPS]
?. [Perfusion and Isolation of Mouse Brain]
Perfuse animals transcardially with physiological saline (10 mL/min) until exudate runs clear (see Note 2).
?. [Perfusion and Isolation of Mouse Brain]
Remove brain and transfer into 50 mL Falcon tube containing  either as whole brain, hemisphere, or brain region.
[HBSS]
?. [Perfusion and Isolation of Mouse Brain]
Finely mince brain tissue with a round-edge blade scalpel to allow fast cooling of the tissue.
?. [Perfusion and Isolation of Mouse Brain]
Spin minced brain tissue for  and aspirate supernatant.
Centrifuge: 400 34, 5 min
?. [Enzymatic Dissociation and Homogenization]
Add enzyme cocktail to the minced whole-brain tissue () or brain hemisphere/brain region () (see Note 3).
10 mL
5 mL
?. [Enzymatic Dissociation and Homogenization]
Incubate for  at  under gentle rotation (see Note 4).
37 °C
?. [Enzymatic Dissociation and Homogenization]
Transfer the digested brain tissue to a 15 mL Dounce homogenizer and dissociate  with 20 passes using the large clearance pestle (see Note 5).
on ice
?. [Enzymatic Dissociation and Homogenization]
Transfer the homogenized brain cell suspension to an equal volume of .
[FBS]
?. [Enzymatic Dissociation and Homogenization]
Centrifuge at  (no brake) and remove the supernatant.
Centrifuge: 400 34, 5 min, 4 10
?. [Myelin-Free Mixed Brain Cell Suspension by Density Gradient]
Resuspend the cell suspension from whole brain in  or brain hemisphere/region in  (see Note 6).
16 mL
[35% Percoll]
?. [Myelin-Free Mixed Brain Cell Suspension by Density Gradient]
Split only the whole brain in 2 × .
8 mL
?. [Myelin-Free Mixed Brain Cell Suspension by Density Gradient]
Carefully overlay each sample with  and leave samples to rest for  (see Note 7).
[1× HBSS]
on ice
?. [Myelin-Free Mixed Brain Cell Suspension by Density Gradient]
Spin samples at  (no brake) and subsequently different layers can be observed (see Fig. 1).
Centrifuge: 800 34, 45 min, 4 10
?. [Myelin-Free Mixed Brain Cell Suspension by Density Gradient]
Aspirate the supernatant including the myelin layer carefully, leaving only the pelleted mixed brain cells.
?. [Myelin-Free Mixed Brain Cell Suspension by Density Gradient]
Wash the cell pellet in  and transfer into a new tube containing .
[1× HBSS]
[1× HBSS]
?. [Myelin-Free Mixed Brain Cell Suspension by Density Gradient]
Spin for  and remove supernatant.
Centrifuge: 400 34, 5 min, 4 10
?. [Immunomagnetic Bead Separation]
Resuspend the cell pellet in  and transfer into a microtube.
[separation buffer ]
?. [Immunomagnetic Bead Separation]
Add  and incubate the cell-bead mix for  at  under gentle rotation (see Note 8).
[anti-CD11b MicroBeads]
4 °C
?. [Immunomagnetic Bead Separation]
Meanwhile place LS single-use columns in magnet and wash through with .
[separation buffer]
?. [Immunomagnetic Bead Separation]
Add  to bead-cell-suspension, apply onto LS column, and collect flow-through (see Note 9).
[separation buffer]
?. [Immunomagnetic Bead Separation]
Wash the columns three times with  (see Note 10).
[separation buffer]
?. [Immunomagnetic Bead Separation]
Remove LS columns from the magnet and place into a 15 mL tube.
?. [Immunomagnetic Bead Separation]
Add  and flush out bead-bound microglial cells by firmly pushing the plunger.
[separation buffer]
?. [Immunomagnetic Bead Separation]
Pellet the cells at .
Centrifuge: 400 34, 5 min, 4 10
?. [Flow Cytometric Analysis]
Resuspend purified microglia in flow cytometry buffer and incubate with  for  at  (see Note 11).
[anti-mouse CD16/CD32]
4 Room temperature
?. [Flow Cytometric Analysis]
Spin at  and remove supernatant.
Centrifuge: 400 34, 5 min
?. [Flow Cytometric Analysis]
Resuspend cells in fluorophore-conjugated antibodies for the protein of interest (here CD11b, CD45, F4/80) for  in the dark at  (see Note 12).
4 Room temperature
?. [Flow Cytometric Analysis]
Wash samples for  at  and resuspend samples in an appropriate volume of flow cytometry buffer.
Centrifuge: 400 34
?. [Immunomagnetic Bead Separation]
Wash the columns with . (1/3)
[separation buffer]
?. [Immunomagnetic Bead Separation]
Wash the columns with . (2/3)
[separation buffer]
?. [Immunomagnetic Bead Separation]
Wash the columns with . (3/3)
[separation buffer]