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Osteoblast Harvest (murine)

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  1. Sacrifice Animals
    Place mice in a bag and fill bag with CO2 - 3-5 minutes, alternatively Dry ice method
  2. Pour Betadine over head
  3. Spray animals with Etoh
  4. Cut Skin sagitally from back of neck towards nose
  5. Remove calvaria with scissors - place in PBS on ice in a petri dish
  6. Scrape soft tissues off calvaria macroscopically and microscopically (total of 2 times)
  7. Place in PBS
  8. Mince with scissors into small pieces
  9. Wash 2-3x in successive dilutions of Betadine:PBS and finally with PBS
  10. Place in Digestive media in a conical tube
    (75 ml DMEM -alpha , 0.150g Dispase II, 0.075 g Collagenase A)
  11. Shake at 37? for 10 minutes - Discard first round of digestion - too many fibroblasts and unwanted cells - collect the subsequent supernatants
  12. Wash and spin (1000 rpm) 3x with PBS to remove enzymes
  13. Plate in Media with serum (10%)


General details
Submitter David Sahar
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protocol details
protocol id 32
protocol views  4569
Added 2003.08.03
Ratings 7 (# 1)
 

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