This protocol outlines a highly reproducible method to functionally and molecularly evaluate paracrine non-canonical Wnt signaling in any cell population of interest. Functional and molecular assessments are performed in the same cell population and can be applied to study any component of the Wnt/PCP pathway. To begin, resuspend the previously pelleted C2C12 cell in 10 milliliters of C2C12 medium.
To dilute the cells at the ratio of 1:20, add 0.5 milliliters of cell suspension in a 15 milliliter tube containing 9.5 milliliters of fresh C2C12 medium and gently mix with a serological pipette using a P1000 pipette, add one milliliter of the diluted cell suspension to each well of a new four chambered well and place it in the incubator. For plating O9-1 cell inserts, resuspend the O9-1 cell pellet in 10 milliliters of O9-1 growth medium. And similar to C2C12 cell dilution, dilute the O9-1 cells at the ratio of 1:20.
Next, place a single permeable insert inside each well of a new four chambered well filled with one milliliter of O9-1 growth medium. Add 300 microliters of the diluted O9-1 cell suspension to each insert and ensure that insert is submerged completely. Then incubate the well at 37 degrees Celsius.
To perform siRNA knockdown in the O9-1 cells, dilute the siRNA and transfection reagent in the reduced serum medium according to the manufacturer's recommendations at the desired concentrations. Gently mix the diluted siRNA and transfection reagent and incubate the mixture at room temperature for seven minutes. After 18 to 24 hours of cell plating, add the siRNA lipid complexes to the O9-1 cell inserts and incubate for approximately 34 to 48 hours.
When the cells reach 70 to 80%confluency, remove the 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 one 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 one 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 nine 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. Begin migration assay termination and well insert system deconstruction by removing the O9-1 cell inserts after nine to 12 hours of the migration period. Carefully aspirate the C2C12 medium.
Add 0.5 milliliters of PBS to the chamber wells and take final images of cells following migration. Carefully aspirate all PBS mixed with medium and remove the plastic chambers from the chamber wells using kit instructions. Leaving the underlying slide containing the C2C12 cells.
For immunostaining, immediately incubate the slide with 4%paraform aldehyde for 10 minutes at room temperature. Then remove paraformaldehyde and wash the slide with 0.1%Triton X-100 containing PBST for 15 minutes at room temperature. After 15 minutes, pour out PBST and wash the slide twice with PBS for 10 minutes each.
Then create a hydrophobic boundary with a hydrophobic pen around the slide to prevent solutions from spilling from the slide preventing disruption of the adherence cells. Next, add approximately 0.5 milliliters of 1%BSA blocking solution to the slide within the hydrophobic boundary and incubate the slide for one hour at room temperature in a humidified slide chamber. At the end of the incubation, remove the blocking solution and add the phalloidin antibody to this slide within the hydrophobic boundary, and incubate at four degrees Celsius overnight.
The next day, place the slide in a slide holder protected from light exposure. Wash the cells thrice with PBS for 10 minutes each at room temperature. Add DAPI-containing mounting medium, mount the slides with glass cover slips and capture the cells using a standard fluorescence microscope.
In the present study, the addition of recombinant Wnt5a to co-culture wells accelerated myoblast migration with complete recovery of some areas by nine hours. The migratory myoblasts in all three conditions exhibited normal migratory cellular morphology, including well-formed and protruding filopodia and lamellipodia and asymmetric polarization of actin cytoskeletal projections. The paracrine effect of NCC-derived Wnt5a on myoblast migration was studied.
Treatment with 50 nanomolar siRNA against Wnt5a reduced Wnt5a gene expression by 64%compared to negative control. The migration of C2C12 mile blasts with significantly reduced following Wnt5a knockdown in NCCs and the addition of exogenous recombinant Wnt5a rescued this migratory deficit and morphological defect in these myoblasts. In order to better understand the signal receiving cell mechanisms in the paracrine model, the ROR2 receptor was knocked down and gene expression was reduced by 54%Knockdown of ROR2 in myoblasts reduces their migratory capacity despite the presence of NCCs.
Exogenous recombinant Wnt5a failed to rescue myoblast migration after ROR2 knockdown suggesting that ROR2 depletion disrupts the ability of myoblasts to receive Wnt5a signals. Culture cells to adequate density scratch with P10 tip only once assemble and unassembled co-culture system with care without disrupting underlying cell. This technique was used as a proof of concept to study a specific molecular signaling axis within the complex Wnt/PCP pathway with relevance for neural crest cell and second heart field biology.
