Using a Macrophage Conditioned Medium to Promote Neuron Extensions

Start with cyclic adenosine monophosphate, or cAMP, a signaling molecule that regulates various cellular functions. Add cAMP to a neuron-macrophage co-culture.

The cAMP molecules from the medium and the growth factors from neurons trigger macrophage activation.

Transfer the insert with activated macrophages to another well containing a macrophage culture medium.

Here, the macrophages release cytokines and chemokines into the medium, forming a conditioned medium or CM.

Centrifuge the CM to pellet the cellular components.

Transfer the supernatant and filter to remove the cellular debris.

Add the collected CM to a neuronal culture. The cytokines and chemokines in the collected CM promote the neuron extensions.

Add chilled paraformaldehyde to fix the cells.

Add a blocking buffer to avoid nonspecific binding of antibodies. Wash to remove the residual buffer.

Apply primary antibodies that target the cytoskeletal proteins of the neurons. Wash to remove unbound antibodies.

Add red-fluorophore-conjugated secondary antibodies that bind to the primary antibodies. Wash to remove unbound antibodies.

Using fluorescence microscopy, visualize the red-colored neurons with 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.