Isolation and Phenotyping of Adult Mouse Microglial Cells
Kathleen Grabert, Barry W. McColl
Abstract
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.
Before start
Prepare Solutions as described in section 'Materials'.
Carry out all procedures at 4°Cand use ice-cold reagents unless otherwise specified ( see Note 1 ). The isolation procedure is summarized in Fig. 1.
Attachments
Steps
Perfusion and Isolation of Mouse Brain
Perfuse animals transcardially with physiological saline (10 mL/min) until exudate runs clear ( see Note 2 ).
Remove brain and transfer into 50 mL Falcon tube containing 10mL either as whole brain, hemisphere, or brain region.
Finely mince brain tissue with a round-edge blade scalpel to allow fast cooling of the tissue.
Spin minced brain tissue for 400x g and aspirate supernatant.
Enzymatic Dissociation and Homogenization
Add enzyme cocktail to the minced whole-brain tissue (10mL) or brain hemisphere/brain region (5mL) ( see Note 3 ).
Incubate for 1h 0m 0s at 37°C under gentle rotation ( see Note 4 ).
Transfer the digested brain tissue to a 15 mL Dounce homogenizer and dissociate 37On ice with 20 passes using the large clearance pestle ( see Note 5 ).
Transfer the homogenized brain cell suspension to an equal volume of 10%.
Centrifuge at 400x g,4°C (no brake) and remove the supernatant.
Myelin-Free Mixed Brain Cell Suspension by Density Gradient
Resuspend the cell suspension from whole brain in 16mL or brain hemisphere/region in 8mL ( see Note 6 ).
Split only the whole brain in 2 × 8mL.
Carefully overlay each sample with 5mL and leave samples to rest for 0h 5m 0s 37On ice ( see Note 7 ).
Spin samples at 800x g,4°C (no brake) and subsequently different layers can be observed ( see Fig. 1).
Aspirate the supernatant including the myelin layer carefully, leaving only the pelleted mixed brain cells.
Wash the cell pellet in 1mL and transfer into a new tube containing 4mL.
Spin for 400x g,4°C and remove supernatant.
Immunomagnetic Bead Separation
Resuspend the cell pellet in 90µL and transfer into a microtube.
Add 10µL and incubate the cell-bead mix for 0h 15m 0s at 4°C under gentle rotation ( see Note 8 ).
Meanwhile place LS single-use columns in magnet and wash through with 3mL.
Add 500µL to bead-cell-suspension, apply onto LS column, and collect flow-through ( see Note 9 ).
Wash the columns three times with 3mL ( see Note 10 ).
Wash the columns with 3mL. (1/3)
Wash the columns with 3mL. (2/3)
Wash the columns with 3mL. (3/3)
Remove LS columns from the magnet and place into a 15 mL tube.
Add 5mL and flush out bead-bound microglial cells by firmly pushing the plunger.
Pellet the cells at 400x g,4°C.
Flow Cytometric Analysis
Resuspend purified microglia in flow cytometry buffer and incubate with 1μg/mL for 0h 20m 0s at 4Room temperature ( see Note 11 ).
Spin at 400x g and remove supernatant.
Resuspend cells in fluorophore-conjugated antibodies for the protein of interest (here CD11b, CD45, F4/80) for 0h 20m 0s in the dark at 4Room temperature ( see Note 12 ).
Wash samples for 0h 5m 0s at 400x g,0h 0m 0s and resuspend samples in an appropriate volume of flow cytometry buffer.
