Material
| Instruments | Solutions |
| 1 Curve Scissor | 50 ml tube 70% Etoh |
| 1 Straight Scissor | Etoh Spray bottle |
| 1 Bone Scraper | 1 bottle of PBS |
| 2 Forceps (1 medium, 1 fine) | 5 10 cm plates |
- Sacrifice Animals
Place mice in a bag and fill bag with CO2 - 3-5 minutes, alternatively Dry ice method - Pour Betadine over head
- Spray animals with Etoh
- Cut Skin sagitally from back of neck towards nose
- Remove calvaria with scissors - place in PBS on ice in a petri dish
- Scrape soft tissues off calvaria macroscopically and microscopically (total of 2 times)
- Place in PBS
- Mince with scissors into small pieces
- Wash 2-3x in successive dilutions of Betadine:PBS and finally with PBS
- Place in Digestive media in a conical tube
(75 ml DMEM -alpha , 0.150g Dispase II, 0.075 g Collagenase A) - Shake at 37º for 10 minutes - Discard first round of digestion - too many fibroblasts and unwanted cells - collect the subsequent supernatants
- Wash and spin (1000 rpm) 3x with PBS to remove enzymes
- Plate in Media with serum (10%)
