Analysis of Immune Cells in Mouse Nervous Tissues Using Flow Cytometry

Take cells isolated from mouse sciatic nerves and dorsal root ganglia or DRGs.               

Incubate with fluorophore-tagged antibodies against CD45 and CD11b, and with biotin-tagged antibodies against MHC class II, to label immune cells.

Incubate with fluorophore-tagged streptavidin that binds to biotin.

Treat with a fixative to stabilize the cellular structure.

Introduce a permeabilization buffer with a fluorescent DNA stain to permeabilize membranes and label the nuclei.

Using a flow cytometer, pass the cells through the laser beam to assess optical and fluorescence characteristics.

Remove aggregates with a higher DNA fluorescence to isolate single cells and detect the fluorescence from the markers.

Identify cells expressing CD45, a characteristic marker of immune cells, confirming their presence.

Most CD45-positive cells in the DRG express CD11b and MHC class II, indicating microglia-like cells, which are absent in the sciatic nerves.

This technique complements imaging for analyzing immune cell heterogeneity within nervous tissues.