Developing a Neuron and Macrophage Coculture In Vitro

Begin with isolated murine dorsal root ganglion neurons in a growth medium.

Seed them onto a polymer-coated multi-well plate to promote neuronal attachment.

Next, take a dissected euthanized mouse and inject ice-cold PBS into its peritoneal cavity.

Gently massage the peritoneum to dislodge macrophages from the peritoneum wall.

Squeeze the mouse to collect the peritoneal fluid and treat it with a lysis buffer to selectively lyse the red blood cells, which appear as contaminants. Centrifuge and resuspend the cells in a culture medium.

Transfer the macrophages to a porous insert positioned within the well containing the cultured neurons.

This co-culture allows neurons and macrophages to be in close proximity.

Treat the co-culture with db-cAMP and incubate to facilitate its entry into the cells. This activates the neurons to secrete the signaling molecules.

These signaling molecules, along with db-cAMP, induce macrophages to adopt a pro-regenerative phenotype and secrete essential factors for neuronal outgrowth.