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This protocol is for isolating single muscle fibers from intact mouse skeletal muscles. It is optimized for use with the Flexor Digitorum Brevis (superficial foot) muscle.
==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.
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.
