The overall goal of this procedure is to observe the effects of the depletion of specific genes of interest on cell migration. This method can help us answer key questions in cancer metastasis, such as cancer cell migration. The main advantage of this technique is that it allows the dynamic cell migration to be monitored in real time.
We first had the idea for this method, when we were observing cell morphology changes upon the patient of TIP60, a tumor suppressor. Begin by seeding one million breast epithelial cells in three milliliters of culture medium per 10 centimeter dish. Add 20 minutes room temperature incubated SRINA mixture drop by drop to the experimental cell culture.
Manually shake both dishes to ensure that the cells are evenly distributed before placing them into a 37 degree Celsius incubator. After six hours, replace the supernatants with eight milliliters of fresh culture medium per dish and return the cultures to the incubator. The next morning, replace the supernatants with three milliliters of fresh culture medium per dish and add a second patch of SIRNA mixture to the experimental cell culture, as just demonstrated, shaking the dish to ensure the reagents are fully mixed before returning the cells to the incubator.
Then replace the supernatants with eight milliliters of fresh culture medium after six hours and incubate the cultures over night. On day three, wash the cells one time with two milliliters of BPS per plate and detach the cells in both plates with one milliliter of tripson EDTA per dish for 20 minutes at 37 degrees Celsius. At the end of the incubation, stop the reaction with four milliliters of complete culture medium per plate.
Resuspend the cells by pipetting and transfer the cell suspensions into individual 15 milliliter tubes. Collect the cells by centrifugation and resuspend the pellets in five milliliters of serum-free medium per tube. After counting, dilute both cell cultures to a 600, 000 cells per milliliter concentration and add 500 microliters of cells from each culture to one well of a 24 well plate to ensure 100%confluency.
When all of the cells have been seeded, gently shake the plate back and forth and side to side to ensure even distribution and place the plate in the cell culture incubator for 24 hours. To check the knock down efficiency of the gene of interest, collect the remaining cells in the collection tubes with another centrifugation and isolate the RNA from the pellets, using a commercial reagent according to the manufacturers instructions for real time quantitative PCR evaluation. The next morning, at least one hour before beginning the live cell imaging, turn on the microscope and temperature control.
Set the temperature to 37 degrees Celsius and carbon dioxide gas supply to 5%When the chamber has reached 37 degrees Celsius, use phase contrast microscopy to confirm that the cells are confluent and fully attached to the bottom of the wells. Then use a 200 microliter pipette tip to gently scratch a straight line across the entire length of the center of each well and gently wash all of the wells five times with serum free medium to remove the detached cells. After the last wash, add one milliliter of serum free medium to each well.
Place the plate on the microscope stage under the 10 power objective and direct the emitted light to the eye pieces instead of the camera. Manually adjust the focus to locate each wound and the cells around it. Then return the emitted light back to the camera and use a live cell observing system to mark the position of the wounds and to set the time conditions.
Image the cells for the appropriate experiment period, then open the first avi video in the appropriate imaging software and open the m track j plug-in. Click on the add tab to add a track for the single cells and choose one cell at the tip of the invasive front and one cell that can migrate on its own. Follow the movement of the chosen cells on the video using mtrackJ to trace their trafficking.
Then click on the measure tab to measure the distance of the cell movement and save the videos with the tracks in avi format. After depletion of TIP60, breast epithelial cells are more mesenchymal compared to control cultured cells. Indeed, TIP60 expression, as well as epithelial cell adhesion molecule expression, decreases significantly after siTIP60 treatment.
On the other hand, the expression of specific epithelial to mesenchymal transition driver genes is increased upon TIP60 depletion. Live imaging of the cell cultures after wound administration demonstrates a faster cell migration of siTIP60 treated cells, compared to siControl cultures, with a significant increase in the percentage of migrating cells observed in response to TIP60 depletion. Further, siControl cells move as a sheet, whereas more single cell movement is observed within the siTIP60 cultures.
After watching this video, you should have a good understanding of how to use a live cell imaging technique to monitor dynamic changes of cell migration.
