Osteoblast Harvest Adult Mice

Material

Instruments	Solutions
1 Curve Scissor	50 ml tube 70% Etoh
1 Straight Scissor	Etoh Spray bottle
1 Bone Scraper1	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 2-3× 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 37℃ for 10 minutes - Discard first round of digestion - too many fibroblasts and unwanted cells - collect the subsequent supernatants

Wash and spin (1000 rpm) 3× with PBS to remove enzymes

Plate in Media with serum (10%)