Material

• 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%)