Basic Protocol for Myotube Culture Derived from P0 Mouse Hindlimb
Need:
Collagen (acid-soluble, Grade A, Calbiochem 234112); 0.5% in 1/100 glacial
acetic acid diluted in sterile water or PBS.
Fine scissors
Dull Dumont forceps
Large forceps
EtOH
Large Pd’s
60 mm Pd’s
30 mm Pd’s
DMEM
DMEM + 0.25% trypsin (2X or 3X crystallized)
DMEM 10F/10H/FPS (DMEM 10/10)
DMEM 10F/FPS (DMEM 10)
Procedure:
1.
Day before culture, collagen coat 35 mm plates. 0.8-1 ml collagen solution per
plate. Swirl to cover the entire bottom of the plate. Place in incubator.
Prepare all media
2.
Day 1:
Culturing:
Immerse mouse pups in EtOH briefly.
Place them in a large Pd and allow much of the EtOH to evaporate.
Decapitate
Quickly remove hindlimbs, place in a 60 mm Pd.
Remove skin and pull off muscles from bones.
Place muscle tissue in a 60 mm Pd and mince into small (2-mm) pieces.
Add to 0.25% trypsin solution for 20 minutes. Place in incubator
Stir gently about every 5 minutes.
Spin down at low speed using desktop centrifuge for 5 minutes.
Gently remove and discard supernatant.
Transfer to a 50 ml conical tube containing 18 ml DMEM 10/10.
Triturate for about 10 minutes (until large chunks break up).
Add about 6 ml of the triturated suspension to 60 mm Pd (3 Pd’s).
Let sit undisturbed in incubator for 20 minutes (fibroblasts stick first).
Transfer what did not stick to 15 ml conical tubes (3 tubes).
Let settle for 3 minutes.
Transfer supernatant to a 50 ml conical tube and count cells.
Adjust volume to about 1 million cells per 1.5 ml medium using DMEM 10/10.
Plating:
Suck out collagen from the 35 mm Pd’s, wash twice with DMEM
Plate about 0.8-1 million cells per 35 mm Pd.
Place in incubator.
3.
Day 3:
48 hours after plating, replace medium with DMEM 10H + 10 M Ara-c.
4.
Day 5:
Replace Ara-c containing medium with DMEM 10H.
5.
Fusion into myotubes should occur beginning 3 DIV.
6.
Myotube cultures ready for coculturing the next day.