Wound Assay to Evaluate the Migratory Capacity of Murine Myoblasts in Co-Culture
Transcription
In response to a wound, secretory cells produce paracrine signaling molecules to activate myoblasts — undifferentiated muscle cells. Activated myoblasts migrate toward the wound site, where they proliferate, causing the wound to heal.
To evaluate the migratory capacity of myoblasts in vitro, take a multiwell plate containing a confluent monolayer of murine myoblasts. Using a pipette tip, scratch the monolayer to create a linear, cell-free patch inside the well, which simulates the wound site.
Carefully supplement the well with the growth medium without disturbing the wound patch.
Take a membranous insert containing an adherent culture of neural crest cells, or NCCs — secretory cells — in a suitable medium. Place the insert inside the well, so that the NCCs at the base are near the wound patch. Incubate the co-culture for the desired duration.
During incubation, the NCCs release signaling molecules that bind to the specific receptors on the myoblasts, initiating a signaling cascade. This signaling facilitates actin reorganization in the myoblasts, and as a result, these cells develop cytoplasmic projections such as lamellipodia and filopodia, at their edges. These projections help the myoblasts migrate toward the wounded area and gradually repopulate, leading to wound healing.
Place the plate under a microscope, and observe the wound healing, the extent of which correlates with the migratory capacity of myoblasts in co-culture.
