Assay of phagocytic function in primary murine microglia

doi:10.1038/protex.2012.017

Method Article

Assay of phagocytic function in primary murine microglia

https://doi.org/10.1038/protex.2012.017

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Here, we describe a method for determining phagocytic function of microglia isolated from mouse brains.

Biological techniques

Neuroscience

Microglia

phagocytosis

in vitro

neural progenitor cells

To determine the phagocytic ability of microglial cells, we prefer to use apoptotic neural cells as targets, since they should most closely approximate natural microglial targets in vivo.

Dissociate neural progenitor cells in 0.1M PBS + 2mg/ml papain (Worthington) at 37º C for 30 m.
Treat dissociated neural progenitor cells (NPC) with UV light for 15-20 minutes; NOTE: this should be done in a thin plastic petri dish; thick plastic (eg. 15 or 50 ml conical tube) will block a significant portion of UV light, leading to suboptimal apoptosis.
Wash NPC in 50 ml conical tube with 40 ml PBS (RT), spin at 1200 RPM, 7 m, RT, low brake. Decant supernatant.
Resuspend NPC in 5 ml RT 0.1M PBS
Add 5 ul sta 5(6)-TAMRA, SE (Invitrogen) to NPC, vortex immediately and let incubate at 37º C in dark for 15 m.
Wash 3X (as in Step 3) with 40 ml ice-cold 0.1M PBS.
Resuspend in 1 ml DMEM/F12 (Invitrogen) + Anti-anti (Invitrogen); count cells using a hemacytometer. Dilute or concentrate to 5 X 10⁶ per ml.
Feed onto microglial cells (previously allowed to adhere to glass cover slips in 24-well plates) in 900 ul DMEM/F12 + 10% fetal bovine serum (Atlanta Biologicals) + Anti-anti at a ratio of 10 NPC:1 microglial cell. NOTE: We typically perform experiments using 5 X 10⁴ microglia per coverslip such that cells are spaced sufficiently to allow later quantification, thus 5 X 10⁵ NPC would be fed on in 100 ul DMEM/F12 + Anti-anti.
Place cells in incubator, at 37º and 5% CO2
At desired timepoints (eg. one, two and five h) remove plates from incubator
In a sterile hood, remove cover slips with forceps, dip 20 X briskly in warm 0.1M PBS (to remove non-phagocytosed NPC), then place cover slips in 1 ml 4% PFA in a 24-well plate. Replace remaining cover slips in incubator until next desired time-point.
Fix cells on cover slips in dark for 20 m. Wash 3X with 0.1M PBS, at RT.
Block cover slips in 10% serum in PBS containing 0.3% Triton X-100 and 0.5% BSA, 1 h, RT in dark.
Incubate with anti-CD11b (eBioscience), 1:100) for 1 h at RT, in dark.
Wash cover slips 3X with 0.1M PBS, RT.
Incubate with desired secondary antibody (1:1000, Invitrogen) for 1 hour at RT.
Wash cover slips 1X with 0.1M PBS, RT.
Incubate in DAPI (1:20,000) 5 m.
Wash cover slips 3X with 0.1M PBS, RT.
Mount cover slips with Aqua-Mount (Lerner Laboratories) on slides. Let dry O/N before imaging.
Typically, a “phagocytic index” can be calculated by dividing the total area of microglial cells (CD11b+) by the total area of labeled NPC. Alternatively, number of phagocytosed targets/cell may be used to quantify phagocytosis.
To check validity of phagocytosed (internal) vs. adhered targets, Z-stacks may be imaged on confocal microscope.

NPC dissociation, 30 m

NPC labeling, 45 m

Incubations, variable (1+ h)

Immunohistochemistry, 3 h

Problem: Little or no phagocytosis observed

Solution 1: NPC were not adequately irradiated; use stronger UV source, extend irradiation time, confirm cell death by dye or antibody-based assay (eg. annexinV, PI).

Solution 2: Insufficient incubation time; extend incubation.

Microglia are highly phagocytic cells. Even after a short period of time, high levels of phagocytosis may be seen, as in Figure 1.

The authors declare there are no competing financial interests

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Assay of phagocytic function in primary murine microglia

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Assay of phagocytic function in primary murine microglia

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Procedure

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Troubleshooting

Anticipated Results

Additional Declarations

Associated Publications

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