Wound Assay to Evaluate the Migratory Capacity of Murine Myoblasts in Co-Culture

1. Wound assay:

(Figure 1 outlines the schematic model of the assay)

1. Allow the O9-1 cell inserts and C2C12 cell chamber wells to adhere and proliferate in the incubator until both cells are at ~70-80% confluency before proceeding with this portion of the protocol. If cells grow >90% confluent, do not proceed with the scratch assay, as cells will merely detach from the well.
2. Warm 1x PBS and C2C12 medium by placing them in a 37 °C water bath.
3. Remove supernatant medium from the C2C12 chamber well and wash the cells once with 1x PBS. Remove the 1x PBS and immediately scratch the cells with a sterile P10 pipette tip.
   1. Pass the sterile P10 pipette tip firmly in a single direction to span the entire length or width of the cell monolayer (e.g., right to left, top to bottom). Be sure to scratch each well containing cells only once.
      NOTE: To optimize scratching results, scratch wells of different experimental conditions at a similar level of confluency. To do this, ensure that each 4-chambered well has cells for each required experimental condition (e.g., well #1 negative control, well #2 positive control). In addition, use a new sterile P10 pipette for each scratch and apply a similar amount of force to the pipette each time. Do not attempt to create more than one scratch in each well.
   2. After scratching, quickly add 1 mL of 1x PBS back into the well using a P1000 pipette tip. Repeat this process for each well that will be scratched.
      NOTE: Given the variability associated with each scratch, it is recommended that multiple wells (n = 3) are used for wound creation in each experimental condition. Work expeditiously as it is critical to minimize the duration for which the cells are without 1x PBS during these steps. After removing 1x PBS from each well, one should not take more than 5 s to generate a wound in each well.
4. After generating a wound and adding 1x PBS back into each well, image the scratch using a brightfield inverted microscope and use this image as the baseline wound size (time 0). To take images, perform the following steps:
   1. Turn on the computer and the microscope (see the Table of Materials) by pressing the power button. Place the chamber slide on the stage and rotate the objective dial to 5x magnification.
   2. Open the imaging software (see the Table of Materials) by double-clicking the software icon on the computer desktop. Click the Camera tab on the software home screen. Click the Live button to visualize the cells on the AxioCam IC tab.
   3. Ensure that the light filter is pulled all the way out to allow light to pass to the camera and computer screen. Manually move and/or rotate the chamber slide to position the wound area in the center of the live image on the AxioCam IC tab.
   4. To take images, click snap to open a new tab next to the AxioCam IC tab that contains the image.
   5. To save this still image, click file on the top left of the software home page | save as | enter the file name in the file name box. Save the figure in Carl Zeiss image (*.czi) format (the default setting), and select desktop on the bar on the left to save the file to the desktop as a .czi file, which can only be opened in the Zen lite 2012 software program.
   6. To save the picture as a .tiff, click file | save as | enter file name in the file name box. Save the figure as tagged image file (*.tiff) by clicking the save as type button and selecting *.tiff from the dropdown menu.
      NOTE: The .tiff format can be opened in any image processing software.
   7. Manually reposition the chamber slide to take 2-3 more images at other points of the wound in the same well.
      NOTE: In total, this will result in 3-4 non-overlapping, high-magnification images of the wound in each well.
5. Remove the 1x PBS from each well and add 1 mL of C2C12 medium.
   NOTE: Be cautious not to pipette too aggressively when removing or adding solutions to the chamber well following wound generation, as this may cause cells to detach from the chamber well. In addition, tilt the chamber well so that aspiration and reintroduction of solutions can be done at the corners of each well to minimize cell monolayer disruption.
6. Assemble the well insert coculture 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.
   NOTE: Do not allow the bottom of the insert to physically touch and mechanically disrupt the underlying C2C12 cells.
7. Allow the cells to migrate for a total of 9-12 h. To determine the optimal migration time, check the cells at 6 h following wound creation, then every 2-3 h thereafter. End the experiment when the cells in control or positive control conditions begin to completely cover the wound.
   NOTE: Given the non-contact nature of the construct, the overlying insert does not need to be removed from the wells when checking interval migration progression. Migratory variability will be observed depending on factors such as the cell types used in this assay, cellular density at the time of wound generation, the width of the wound created, and experimental conditions of manipulated cells (e.g., gene knockdown, recombinant protein addition). Concentrations of these reagents should be determined experimentally with guidance from manufacturer recommendations.

Figure 1: Schematic model of the assay. Step 1 includes the in vitro expansion of C2C12 myoblasts and NCCs using STO feeder cells. Step 2 involves the plating of NCCs and C2C12 cells in the coculture system. Step 3 includes the wound assay performed in underlying C2C12 cells to evaluate cellular migratory capacity. Step 4 involves immunostaining for phalloidin to evaluate cytological architecture and morphology of migrated cells. Abbreviations: NCCs = neural crest cells; Ab = antibody.