Single fiber dissociation of intact rodent muscles
Materials
1. Dissection tools
2. Incubation medium (warm to 37 deg C)
- D-MEM (high glucose), containing:
- 2% FBS
- 5% PSG
- 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
- 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).
- 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.
References
This protocol is a modified method of:
- Cho et al., (2016) FASEB J 30(2):674-87 http://www.fasebj.org/content/30/2/674.full
- Spangenburg et al. http://www.seahorsebio.com/resources/tech-writing/techbrief-intact-muscle-fiber.pdf