Gebruik te maken van op maat ontworpen galvanotaxis Chambers naar Study Directional Migratie van Prostaat Cellen

The overall goal of this procedure is to perform Galvan Axxis, an assay to assess the directional migration of cells in an applied electric field. This is accomplished by first assembling and then seeding a Galvan axxis chamber with the cells of interest. In the second step, the chamber is sealed and an electric field is applied for time-lapse imaging.

In the final step, the migration speed of individual cells and the angle the relative to the electric field is tracked. Ultimately cosign. The can be used to show how the cells respond to the electric field and whether they migrate directionally to the cathode.

The main advantage of this technique are the easy cell retrieval after given Texas for secondary analysis and the ease with which the migration can be filmed at high magnification and with fluorescently labeled cells. Visualization of this method is critical as a chamber assembling steps are difficult to learn without demonstration To assemble the bottom chambers. Begin by wiping a plastic galvan axxis chamber with two propanol.

Then use a syringe to apply marine grade silicon sealer around the circular openings on the bottom side of the chamber. Arrange a large cover glass over the sealant and press it into place with the end of a cotton applicator, wiping off the excess glue with the cotton swab. Then use a diamond point marker to cut a small cover glass to make six by 25 millimeter spaces and flip over the chamber.

Next, use a syringe to apply two stripes of silicone, creating a 10 by 25 millimeter channel surface for cell attachment between the two circular openings on the bottom glass. Then glue two glass spaces between the circular openings, pushing down the spaces with a new cotton applicator and wiping off the excess glue as just demonstrated. Try the chamber for 24 hours until the silicone glue is cured.

The next day, soak the chamber in distilled water overnight to remove the acetic acid residue from the glue before seeding cells onto the chamber. Dry and wipe the chambers with two propanol as just demonstrated. Wash them with sterile PB S3 times and then check the liquid flow between the two circular openings in the chambers.

After confirming the chambers are in good working condition, enclose them in sterile culture dishes and place the chambers at 37 degrees Celsius for 15 minutes. While the chambers are e equilibrating, detach the prostate cells from their culture dish with five milliliters of trypsin EDTA at 37 degrees Celsius. After five minutes, neutralize the reaction with five milliliters of 10%FBS.

In PBS, transfer the detached cells to 15 milliliter tubes and then centrifuge for five minutes. At 200 times, G, aspirate the medium and then resuspend the pellet in one milliliter of medium. Then after counting the cells, adjust the cell concentration to eight by 10 to the four cells per milliliter with culture medium and seed.

350 microliters of the cell suspension onto each chamber. Incubate the cells in the chamber's overnight in a culture dish with a wet Kim wipe at 37 degrees Celsius and 5%carbon dioxide. The next day, begin assembly of the top chambers by first warming up 10 milliliters of culture, medium to 37 degrees Celsius for each galvan AXS chamber.

While the media is warming, transfer a Galvan axxis chamber from the incubator onto a 37 degree Celsius warming plate and rinse the chamber with culture medium. To remove the unattached cells, leave 400 microliters of medium in the chamber and then use a syringe to apply high vacuum grease on top of both of the glass spaces. Seal the chamber with a cover glass and a cotton applicator, and then dry the glass surface.

Next, apply the high vacuum grease with a syringe to seal the gap between the cover glass and the chamber. And then add the culture medium to the inner reservoirs. Remove bubbles and check the liquid flow between the reservoirs.

To make the agar bridges cut a pair of two inch PVC tubes, flip them and then place the pieces in a 100 milliliter beaker. Next, add 200 milligrams of bact toe agar into a 50 milliliter flask with 10 milliliters of culture medium to make a 2%agar gel. After microwaving for 30 seconds, use a transfer pipette to load the agar gel into the plastic tubing and leave the agar bridges at room temperature for 10 minutes to solidify the agar.

Then add two milliliters of culture medium to the outer reservoirs of the gal chamber, and insert the agar bridges into the inner and outer medium reservoirs to conduct the current to perform time-lapse imaging of the migration. Begin by transferring the Galvan chamber into a 37 degrees Celsius environmental chamber on the microscope stage, securing the chamber with tape and focusing on the cells under a 10 x objective. Next, insert the Galvan Axxis electrodes into the outer reservoirs with the cathode on the right side, securing the wire and the electrodes with tape.

Then switch on the power box to apply an electric field to the chamber, and use a voltage meter to measure the output voltage across the chamber to reach 2.5 volts for the 25 millimeter long chamber. Now select 10 points across the chamber for generating 10 time lapse movies, and set the acquisition conditions to 10 minutes intervals for two hours. Then start filming and adjust the output current as needed to maintain the field strength during the experiment.

At the end of the experiment, remove the chamber from the stage and fix the cells in 95%alcohol. Then break open the chamber. The cover slip can be saved for later.

Staining and imaging. Clean the chamber for future years. To quantify the Galvan Axxis, rotate the time-lapse movies to orient the cathode to the top of the images.

Export the movies to the cell tracking software and manually track the XY position of 10 to 20 cells at each time point from each movie, the migration distances and the angles relative to the north south direction, the same orientation of the electric field will be calculated in the cell tracking software. Finally, save the measurements and import the data to a database application to calculate the combined results. In this representative Galvan Axxis experiment to lines of prostate cells were determined to migrate at similar speeds over the course of two hours.

However, the directionality to the electric field was 0.7 plus minus 0.3 for the PRNS one one line, and 0.2 plus minus 0.8 for the PNT two line, indicating a significant difference in the Galvan axis of these two cell lines, suggesting their cellular signaling mechanisms direct differential migration responses to the electric field After the development of the Govan Texas method. This technique help researchers to explore the mechanisms of directional cell migration in the electro field.