Single fiber dissociation of intact rodent muscles

Materials

1. Dissection tools

2. Incubation medium (warm to 37 deg C)

3. Dissociation medium (warm to 37 deg C)

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

Anesthetize & CD mouse
Dissect out muscle/muscles of interest
Place into culture dish containing 2 mL of incubation medium & allow to recover in incubator (15-30 min)
Carefully remove the medium & replace with 4 mL of dissociation medium
Incubate for 2 hr
Prepare fresh culture dishes containing 2-4 mL incubation medium
Using a Pasteur pipette, carefully remove the muscle & place in new culture dish. Take care not to transfer too much of the dissociation medium.
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.
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.
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.
Let muscle fibers recover overnight. They are now ready for use experimentally.

This protocol is a modified method of: