Set aside three hours to conduct this protocol. Once you are experienced, this protocol should take anywhere between one and a half and two hours to complete depending upon the number of embryos dissected. This protocol describes how to take a 9.5 days post cort embryo cut at the mid otic plaque code and som might four to isolate tissue containing the vagal neural tube.Cutting.
At so mite 16 and 22 will isolate tissue containing the trunk neural tube. Vagal neural crest migrates from the vagal neural tube. While trunk neural crest migrates from the trunk neural tube, the dissected tissue will be enzymatically digested in a solution of collagenase dys, space washed and non neural epiderm and mesoderm will be dissected away.
The isolated neural Tube will be washed twice more and then plated in self-renew medium. I'm Elise Graph of the Bosky Laboratory At Vanderbilt University. Today I will be demonstrating how to isolate the neural crest from the neural tube in explan culture.
This technique will allow you to study the characteristics of the neural crest In vitro. Let's get started. Begin by diluting 100 microliters of one mg per mil fibronectin into 3.2 milliliters of sterile DPBS.
Place enough of the 30 microgram per milliliter fibronectin solution to cover the bottom of each well of a four well plate. Gently tap the corners of the four well plate to ensure every well is evenly covered. Let the plate sit aside while you make and filter your media.
The plate should sit at least 15 minutes. Collect the following reagents To prepare your media. The media are made up in a biosafety cabinet fresh before use.
Be sure to use sterile technique at all times. Remove the fiber nin from the four well dish and allow it to dry with the lid off in the back of the hood. It takes about 15 minutes for the fiber nin to dry.
Once the plate is dry, add 500 microliters of self-renew medium to each. Well place the plate of 37 degrees in a humidified 5%carbon dioxide incubator immediately before starting the Dissection mix 50 microliters collagenase disc space into five milliliters of DPBS. Be sure to add all of the collagenase dispa to ensure an efficient digestion.
Use a 0.2 micron syringe filter to filter the collagenase dispa solution into three wells of a 12 well plate. Each well should receive one third of the five milliliter solution Pipette approximately one milliliter of wash medium into each of the remaining wells. Place the plate on ice until you are ready to digest your isolated tissue.
Next, cut the tip off a P 20 pipette tip just below the beveled edge with a sterile razor blade. Dip the tip in wash medium so embryos and pieces of isolated tissue. Do not stick to the tips while being transferred between solutions.
Do the same to The tip of a P 1000 from a timed mating. Remove the uterus from the dam. Complete the dissection in sterile DPBS.
Sterilize your dissection tools using any method such as heat sterilization. If the DPBS becomes murky, move the embryo with the cutoff P 1000 into a fresh dish of sterile DPBS beginning at the cut tissue. Gently tease the uterine tissue away from the decidua.
Next, gently remove the embryo from within the decidua. The location of the embryo is indicated here with a cartoon. After isolating a 9.5 days post coum embryo, gently remove the yolk sack with insulin needles.
If genotyping is a necessary part of your protocol, wash the yolk sack and any remaining embryonic tissue in clean, sterile DPBS. Place the tissue in a 1.5 milliliter tube and store the tube at negative 80 degrees Celsius until you are ready to isolate DNA. Orient yourself to the anatomy of the embryo.
You'll be cutting at the mid OTIC PLA code in SOMITES four 16 and 22. You will need to adjust the focus of your microscope In order to count the somites. Make the first cut at the mid Otic PLA code.
Use the needles like the blades of scissors without damaging the rest of the neural tube. Make the second cut after so mite four. Next cut to so mite 15 and cut immediately after it.
Make the final cut After so mite 22 or the last, so mite. If the embryo is developmentally earlier. This tissue contains the trunk neural tube to isolate the tissue containing the vagal neural tube.
Cut the heart away from the tissue between the mid otic plateau and the fourth so mite. Notice the difference in size between the pieces of isolated tissue. The piece containing the vagal neural tube is broader than the other.
This physical distinction allows you to process the two pieces together. Use the cutoff P 20 to transfer the neural tube to collagenase dis space. The remaining tissue can be kept for genotyping.
As stated previously, place the pieces of isolated tissue in one of the wells with collagenase dis space. Now at room temperature. Let it sit for 10 minutes.
During the 10 minute incubation, you can begin dissecting the Next embryo. After 10 minutes, Remove the isolated tissue from the collagenase dis space and wash by pipetting up and down in wash medium, place the digested tissue in DPBS. This is what the tissue should look Like after the digestion.
The next step involves removing the non neural derm and mesoderm. First, the non neural derm is lifted away from the neural tube. Next, the mesoderm can be removed from the neural tube.
To demonstrate the removal of non neural derm and mesoderm, we will focus on the vagal neural tube. The same process can be done with the trunk neural tube. Begin by holding down the non neural tissue with one needle.
Use the other to gently remove the non Neural derm. Next, gently remove the somites adjacent to the neural tube to remove the remains Of any non neural tissue. Gently iterate with the cutoff P 20 pipette tip.
Wash the neural tube twice with Wash medium. Place the neural tube into self-renewal medium, and then plate into a prepared four. Well Dish.
Put the four well plate into an hypoxia chamber. Be sure the chamber is sealed. Attach an oxygen analyzer And turn on the source of mixed gas.
Use a mix of 1%oxygen, 6%carbon dioxide, and 93%nitrogen. Charge the hypoxia chamber until it is at 3%oxygen. Turn off the mixed gas and place the hypoxia chamber at 37 degrees Celsius.
24 hours later. Look at the neural tube explan. Neural crest cells should have migrated from the neural tube outlined with the solid line to prevent contamination with non neural crest tissue cut around the outside of the neural tube with an insulin needle.
Next, remove the neural tube from the explan and any remaining non neural crest cells. Replace the medium with fresh self-renewal medium and return the plates to the hypoxia chamber. Charge it and place it at 37 Degrees Celsius.
After 24 hours in culture, The neural crest will migrate away from the neural tube. The extent of the outgrowth is shown with a dashed line. This neural crest outgrowth highlighted by arrows is approximately 96%pure and the cells express P 75 SOX 10 and Fox D three.
Experiments that can be conducted with these cultured crest cells include, but are not limited to the following. Sometimes less ideal cultures will not yield efficient outgrowths if this occurs. Double check hypoxic conditions, fibronectin concentrations, and the amount of time the tissue was digested in collagenase DYS Space today I have demonstrated the basics behind culturing the neural crest from the neural tube.
Good luck with your experiments.
