Achilles Tendon Dissociation

1.    Scrape off residual muscle and connective tissue with a scalpel

2.    Rinse 3-4 times with sterile DPBS to remove fur and debris

3.    Place in collagenase solution (~20 mL for 2 tendons, 30 mL for 4 tendons)

a.             1 mg/mL type I collagenase

b.             DEM or F-12 containing 20% FBS

c.              1% ABAM

d.             Sterile filter with steriflip

4.    Put in 37¼C water bath for 17 - 48 hours

5.    Shake vigorously at the end of the incubation (tendon should disintegrate)

6.    Centrifuge for 5 min at 2500 rpm

7.    Aspirate supernatant

8.    Resuspend in 10mL GM per 2 tendons

9.    Plate on 2 X 15cm dish (T75 flask) per 2 tendons

Growth Media (GM)

80% F12 HAMS

20% FBS

1% ABAM

 Differentiation Media (DM)

90% F12 HAMS

10% Heat inactivated FBS

1% ABAM

Supplement fresh before each feeding with:

50µM L-ascorbic acid 2-phosphate (A8960)

50µM proline