Preparation of Collagen-Coated Compartmented Culture Dishes for Neuronal Cell Culture
Transkript
To assemble compartmented culture dishes, dilute collagen stock solution to 500 micrograms per milliliter in sterile distilled water and mix thoroughly. With a sterile transfer pipette, fill a 35-millimeter culture dish with 2 milliliters of the diluted collagen solution. Then, slightly tilt the dish, and remove the solution, leaving a thin film of collagen behind. Dispense the solution into the next 35-millimeter dish. Repeat this process, adding more collagen solution as needed until all dishes have been coated.
To polymerize the collagen in a laminar flow hood, wet gauze pads with 3 milliliters of concentrated ammonium hydroxide, and cover the trays for 15 minutes. Remove gauze pads and allow the 35-millimeter dishes to dry. Next, use a metal file to file off the point of an 18-gauge needle to make a blunt tip. Soak the needle in 70% ethanol to sterilize. Apply silicone grease to the bottom of the compartmented chamber. Ensure that grease is placed neatly and overlaps at all corners.
This technique can be very challenging, so we suggest that you take extra time to practice applying the correct amount of grease before setting up the compartmented chambers in order to ensure axonal growth. It is also very important to work slowly. Set up more chambers than you think you will need, so you have extras should something go wrong.
Remove the lid from a 35-millimeter dish. Invert the dish, and place the scratches over the chamber. Tap down on the bottom of the dish gently with a pair of forceps.
When applying the grease chambers to the dish, do not tap too hard. Otherwise, the axons will not be able to grow underneath into the adjacent chamber.
Use the hemostatic forceps to gently flip the dish over. Release the forceps. Place a mound of grease at the base of the center compartment. Fill each chamber with supplemented neuronal basal medium. Check for leaks, and seal leaks with silicone grease as needed.
