修正Zigmondチャンバーアッセイを使用してトランク神経冠細胞移動の解析

The overall aim of this procedure is to evaluate the ability of a molecule to elicit chemotaxis and other migratory responses in explanted trunk neural crest cells. This is accomplished by first isolating trunk level neural tubes between roughly eight and 15 somites in length and culturing each of the neural tubes overnight on separate fibronectin coated cover slips to allow for neural crest emigration. Next, an optimal neural crest culture is selected.

The neural tube is removed from the culture and the cover slip containing the culture is mounted onto a modified zigmund chamber so that the straightest border of the culture is parallel to the vector of the molecular gradient to be applied across the culture. The third step is to generate a molecular gradient across the culture and then film the migration of the peripheral neural crest cells along the previously selected straight border of the culture. The final step is to track and analyze the migration of the peripheral neural crest cells positioned along the selected border of the culture using Image J software.

Ultimately, it can be determined whether the selected molecule can alter cell directionality or other migratory characteristics such as speed through the analysis of the cell trajectory data obtained. The main advantage of this technique over existing techniques like the Boyden chamber assay, is that at a very low cost. This method can be used to analyze the migration of already polarized cells on the periphery of primary explan cultures using time-lapse imaging.

After incubating chick eggs for 56 hours at 38 degrees Celsius, remove the eggs from the incubator, mildly spray them with 70%ethanol, and then allow the eggs to dry while the eggs are drying. Fill one sterile plastic Petri dish with Chick ringer solution and UV sterilize a glass tray. Fill a five centimeter glass Petri dish with dis displays.

Now break the eggs open into the UV sterilized glass tray. Extract each embryo from its yolk by first cutting around the embryo's blood islands with curved scissors. Then with blunt forceps, pick up the embryo by its extra embryonic membrane and place the embryo in the prepared Petri dish containing ringers.

Next, select about nine embryos that are between hamburger and Hamilton. Stages 15 to 17 with a tungsten needle cuts off any embryonic tissues anterior to so mite 10 and remove all cordal embryonic tissues starting from around the fifth most newly formed so mite. Then trim off extra embryonic membranes to about two millimeters from the embryo.

Place the isolated embryo trunks in dis bays and incubate them for one hour and 15 minutes at 37 degrees Celsius and 5%carbon dioxide while the embryo trunks are incubating. Prepare six cover slips for culturing. First by rinsing them in 70%ethanol and allowing them to dry.

Then using a lab marker, draw a circle in the center of each cover slip that is about one centimeter in diameter for later identification of the fibrin connecting coat on the same face of each cover slip. Write an asymmetrical word or symbol outside the drawn circle to facilitate the identification of the top and bottom of the cover slip. Now place each cover slip in a separate 40 by 10 millimeter sterile dish with the labeled side facing down, and allow the dish to sit open under a germicidal UV lamp for 10 minutes.

Then apply 60 microliters of fibronectin into the unmarked surface of the cover slip within the one centimeter circle, making sure that the entire area of the circle is coated. Place the dishes in the incubator at 37 degrees Celsius for 30 minutes. After the incubation period, carefully aspirate the fi nin from each cover slip.

Next, add 250 microliters of culture medium to the FI nin coated area. The cover slip, and then incubate the cover slips again until the neural tubes have been isolated. While the cover slips are incubating, fill a five centimeter glass petri dish with L 15 medium.

Now transfer all the incubated embryo trunks to the prepared Petri dish. Use fine forceps and a sharp tongues and needle to dissect out the neural tube by carefully slicing along the border of the neural tube and the somites with the needle. Remove the cover slips from the incubator.

Select six of the longest and straightest neural tubes, and then using a micro pipette tip primed with culture medium, transfer one neural tube onto each of six cover slips. Ensure each neural tube is within the fibronectin coated area of its respective cover slip using a micro pipette and incubate the overnight at 37 degrees Celsius and 5%carbon dioxide. Next place at least two milliliters of culture medium without serum into a sterile 15 milliliter centrifuge tube.

Leave the calf slightly unscrewed while incubating the tube overnight at 37 degrees Celsius and 5%carbon dioxide to allow the pH of the medium to adjust. After overnight culture, select the three best neural crest cell cultures that have at least one long straight edge outta the three cultures. Choose one for loading the first chamber and return the others to the incubator for later use.

Then using our cotton swab, apply a thin even layer of petroleum jelly to the areas surrounding the reservoirs and bridge of one modified sigmund chamber. Next with a tungsten needle, gently remove the neural tube from the cover slip containing the selected neural crest cell culture, leaving the surrounded neural crest cells attached to the fibronectin coat. Mark the dish with a lab marker in order to remember the orientation of the straightest border of the neural crest cell culture.

Next, place a few drops of the pre incubated medium onto the bridge of the modified zigmund chamber. Then pick up the cover slip with fine forceps. Dab the edge of the cover slip against a Kim wipe to remove most of the old culture medium, and immediately place the cover slip on the modified zigmund chamber so that the straight neural crest cell border to be filmed is centered over the length of the bridge and roughly perpendicular to the bridge reservoir border.

Using an inverted microscope, move the straight neural crest cell border to the side of the bridge closest to the reservoir. That will contain the suspected chemo tact and more finely align the straight neuro crest cell border perpendicular to the bridge reservoir border. Now carefully but securely press the cover, slip into the petroleum jelly present on the zigmund chamber, making sure it is completely sealed onto the chamber.

Then place additional petroleum jelly along the edge of the cover slip to further ensure that it will be airtight fine. Adjust the angle of the neural crest cell border again to correct for any movement during the ceiling process. Next, load roughly 300 microliters of pre incubated medium into a one milliliter syringe.

With a 25 gauge by 1.5 inch attached needle. Inject the medium into the reservoir that will not contain any chemo tact until full. Being careful not to generate any bubbles in the reservoir.

Then plug the reservoir on both sides with a sufficient amount of petroleum jelly prior to loading the next reservoir. Now load a second syringe with 300 microliter of pre incubated medium containing the desired chemo tact. Then inject the medium into the opposite reservoir in the same way.

Again, carefully sealing the reservoir with petroleum jelly. After incubate the loaded Sigmund chamber at 37 degrees Celsius for one hour prior to filming. Then while incubating at roughly 37 degrees Celsius image, the straightest border of the neural crest cell culture for three hours at 92nd intervals for controls, load zigmund chambers containing each of the two other best neural crest cell cultures as just shown.

Using the appropriate control treatments for filling the reservoirs and priming the bridge. Use the image J manual trackin plugin to track the migration of peripheral neural crest cells along the straight border of the culture. Use the chemotaxis and migration tool plugin to analyze various parameters of the migratory trajectories obtained.

Elongated trunk neural crest cell cultures were prepared by overnight culturing of neural tubes and resultant neural crest cell cultures with at least one long straight border was selected for experimentation. The longest straight border of one selected culture was then positioned perpendicular to the bridge reservoir border, and therefore parallel to the vector of the future applied gradient. The reservoir that did not contain the suspected chemo attractant represented here with a minus sign was loaded first and sealed.

Then the other reservoir was loaded with the suspected chemo attractant represented with a plus sign and sealed peripheral neural crest cells along the previously selected border were then imaged and tracked using the manual tracking plugin for image J.Numerous migratory characteristics in response to the applied gradient can be assessed based on the tracking data obtained as illustrated with this graph. For instance, a chemotaxis index can be derived by dividing a cells displacement along the x axis by the total distance it has migrated. The attractive response of all the cells tracked is demonstrated by the cell trajectory plot shown here.

Each red track is the trajectory of a cell that migrated toward the reservoir loaded with a suspected chemo attractant. In this figure, there is a substantially greater amount of red tracks compared to black. In another test, the ability of a modified sigmund chamber to maintain a molecular gradient was validated.

An Alexa Fluor 4 88 IGM conjugate was added to the left reservoir and the fluorescence intensity across the bridge was measured at various times. The gradient remained for at least 26 hours After watching video, you should have a good understanding of how to analyze a molecule's ability to elicit chemotaxis and other migratory behaviors in explanted trunk neural crest cell cultures using a modified zigmund chamber assay.