Using a Macrophage Conditioned Medium to Promote Neuron Extensions

Four hours after the neuron macrophage have been co-cultured, add 2 microliters of 100 micromolar db-cAMP solution to the neuron macrophage co-cultures. After 24 hours, fill an empty well with 1 milliliter of macrophage culture medium in the same 6-well plate.

Transfer the cell culture insert in the neuron macrophage co-culture to the empty well with macrophage culture medium. After 72 hours, centrifuge the macrophage-conditioned medium at 239 g for five minutes to remove the cellular components. Pass the supernatant through a 0.2-micrometer filter to remove any remaining cellular debris.

In this procedure, precoat an 8-well chamber slide with poly D-lysine and laminin. Then, obtain the associated adult DRG neurons in neurobasal medium, supplemented with B 27. Plate 5 x 10⁴ cells per well onto a precoated 8-well chamber slide.

Next, place the chamber slide in a 37 degrees Celsius incubator for two hours, allowing the cells to attach to the bottom. Then, replace the culture medium with a thawed conditioned medium that is preheated at 37 degrees Celsius. 15 hours after the initial plating, remove the medium and wash the cells with PBS once.

Then, add 200 microliters of ice-cold 4% paraformaldehyde solution to the wells and incubate the cells with the paraformaldehyde solution for 20 minutes at 4 degrees Celsius. Subsequently, incubate the fixed cells with primary antibody solution, diluted with 10% normal goat serum for four hours at room temperature or overnight at 4 degrees Celsius. Afterward, take images using a fluorescence microscope to visualize neurite outgrowth.