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Osteoblast Harvest Adult Mice 7/17/02
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