Encyclopedia of Experiments: Biology
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Transcript
When the cells reach 70% to 80% confluence, remove the supernatant medium from the C2C12 chamber well, and wash the cells once with PBS.
After removing the PBS, begin scratching the cells by passing a sterile P10 pipette tip firmly, in a single direction to span the entire length or width of the cell monolayer. Once the cells are scratched, quickly add 1 milliliter of PBS.
Next, turn on the computer and the microscope. Place the chamber slide on the stage, and rotate the objective dial to 5X magnification. Open the imaging software, click the "Camera" tab, and then click the "Live" button to visualize the cells on the "AxioCam IC" tab.
Ensure that the light filter is pulled all the way out to allow light to pass to the camera and computer screen. Manually move or rotate the chamber slide to position the wound area in the center of the live image on the "AxioCam IC" tab. To take images, click "Snap" to open a "New" tab next to the "AxioCam IC" tab that contains the image.
To save this still image, click on the "File," select "Save As," select "Desktop" on the bar on the left to save the file to the desktop. Enter the "File name" in the file-name box, and save the figure in ".czi" file format.
To save the picture as a ".tiff," click "File," select "Save As," enter the "File name" in the file-name box, click the "Save-As-Type" button, and select "*.tiff" from the dropdown menu.
Manually reposition the chamber slide to take two to three more images at other points of the wound in the same well. After imaging, remove the PBS from each well, and add 1 milliliter of C2C12 medium.
Assemble the well-insert co-culture system by manually placing the inserts containing the O9-1 cells in each well of the chamber well. Gently, push the inserts down into the well such, that the bottom of the insert sits just above the underlying C2C12 cells. Return the well-insert constructs to the incubator, and allow the cells to migrate for a total of 9 to 12 hours.
To determine the optimal migration time, check the cells at six hours following wound creation, then, every two to three hours after that. End the experiment when the cells in control conditions completely cover the wound.
