Treatment
Collagen Gel Methods
1. Sterilize the MED probe with 70 % ethanol (DO NOT AUTOCLAVE).
2. Dry up the MED probe in a clean bench and further sterilize it with UV radiation for 15 min.
3. Place the MED probe in a refrigerator for at least 1 hr.
4. Take out the MED probe from the refrigerator and pour the collagen solution (see below) into the MED probe until the collagen solution completely covers the probe surface surrounded by the ring, then immediately remove the collagen solution with glass pipette. The collagen solution can be used for the coating of other probes.
5. Incubate the MED probe in a CO2 incubator for 30 min for collagen to form gel matrix.
6. Rinse the MED probe three times with sterile distilled water.
7. Pour culture medium into the probe and keep it in a CO2 incubator until use (can be stored up to one week).
NOTE: All steps should be carried out aseptically for culture preparation. This process should be done at least 8 hrs before placing a slice on the probe to make sure the coated probe is not contaminated.
(Collagen solution preparation)
All steps should be carried out aseptically at 4 C
1. Add 1 ml of 10x DMEM/F-12 mixture medium to 8 ml of collagen gel solution (in 0.01 N Hydrochloric acid, pH 3.0) to Collagen gel solution (Nitta gelatin ; Cellmatrix Type I-A) and stir gently.
2. Add 1 ml of preparation buffer (200 mM HEPES in 0.08 N NaOH) to mixture and stir gently.
3. Place the mixture in a refrigerator for 30 min. to remove bubbles (this step may be skipped).
