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Osteoblast Harvest(Adult Mice)

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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%)

 

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