Encyclopedia of Experiments: Immunology
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Transcript
The dorsal root ganglion, or DRG, are a cluster of neuronal cell bodies along the spinal cord that carry sensory information from the peripheral to the central nervous system. Peripheral nerve injury causes macrophage accumulation in the DRG and the development of neuropathic pain.
To isolate viable macrophages using an enzyme-free method, take freshly dissected mouse DRG tissue in a homogenizer filled with ice-cold buffer to limit the cellular stress. Using a pestle, mechanically homogenize the tissue to release the cells.
Filter through a cell strainer to remove cell clumps and obtain a single-cell suspension in the flow-through. The tissue homogenate contains neurons and non-neuronal cells, which include macrophages and free-floating myelin — the insulating sheath of neurons.
Add the filtered homogenate into a tube containing a density gradient medium, and centrifuge. Under centrifugal force, the viable cells with higher buoyant density settle at the bottom of the tube to form a pellet, while the myelin remains suspended. Discard the supernatant.
Resuspend the cells in a buffer containing fluorophore-conjugated antibodies against macrophage-specific cell-surface receptors. Incubate for the required duration. The antibody binds to its receptor on the macrophage cells, thus, fluorescently labeling the cell surface. Wash to remove unbound antibodies.
Analyze the cells using a flow cytometer. An antibody-specific fluorescence signal indicates the presence of macrophages in the sample.
