Single fiber dissociation of intact rodent muscles

Materials

1. Dissection tools

2. Incubation medium (warm to 37 deg C)

• D-MEM (high glucose), containing:

1. 2% FBS
2. 5% PSG
3. 1mM Na Pyruvate

3. Dissociation medium (warm to 37 deg C)

• Incubation medium plus 4 mg/mL type-2 Collagenase

4. 35 mm cell culture dishes (or 6-well plates)

5. Glass Pasteur pipettes (1 mm bore) & pipette bulb

6. Incubator (37 deg C, 5% CO2)

7. 5 mL tubes

Protocol

1. Anesthetize & CD mouse
2. Dissect out muscle/muscles of interest
3. Place into culture dish containing 2 mL of incubation medium & allow to recover in incubator (15-30 min)
4. Carefully remove the medium & replace with 4 mL of dissociation medium
5. Incubate for 2 hr
6. Prepare fresh culture dishes containing 2-4 mL incubation medium
7. Using a Pasteur pipette, carefully remove the muscle & place in new culture dish. Take care not to transfer too much of the dissociation medium.
8. Gently pipette the muscle up & down to mechanically separate the muscle fibers. After 10 passes, return the remaining muscle bundle to the dissociation medium & return to the incubator for 20 min.
9. Repeat last two steps. You may need to repeat this 2-3 times before the connective tissue is digested enough to yield ~80-90% of the muscle as single fibers.
10. Remove debris (tendons, blood vessels, nerves, undigested muscle) using fine forceps & allow fibers to recover in the incubator for 30 min (overnight if additional cleaning is not required).
    1. Optional additional cleaning step: Transfer fibers in incubation medium to 5 mL tubes & allow to settle via gravity sedimentation (approx. 20 min). Carefully remove medium & replace with fresh incubation medium. Repeat once more, before placing cleaned fibers into fresh culture dishes.
11. Let muscle fibers recover overnight. They are now ready for use experimentally.