Using a Macrophage Conditioned Medium to Promote Neuron Extensions
Transcrição
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.
