Prepration of primary myoblast by pre-plating
Prepration of primary myoblast by pre-plating
Material
- Two mice
- PBS
- Collagenase II sol (2 mg/ml collagenase II in DMEM/10% FBS/PS, -20C)
- DMEM (DMEM/10% FBS/PS)
- Growth medium for myoblast (Ham's/F10, 20% FBS, 2 ng/ml bFGF, PS)
- Fungizone (Amphotericin B) (250 ug/ml, Gibco 15290-018, -20C)
- 12 ml syringe with 18G needle
- 70 um Cell Strainer (Fisher Scientific, 22-363-548)
Day 1
- Euthanize mouse and peal skin.
- Dissect pectoral, brachial, leg, and abdominal muscle.
- Wash muscle by PBS on 10 cm petri dish.
- Eliminate fat, tendon, and vessel.
- Mince muscle in 10 ml collagenase II sol on 6 cm petri dish.
- Incubate at 37C for 1 h.
- Suspend by 12 ml syringe with 18G needle.
- Incubate at 37C for 15 min.
- Put into 25 ml DMEM in 50 ml tube.
- Filtrate by 70 um Cell Strainer into 50 ml tube.
- Keep tube on ice in following procedure.
- Centrifuge at 2000 rpm at 10C for 5 min.
- Suspend in 21 ml growth medium with 250 ng/ml Fungizone (1/1000 of stock).
- Seed on three non-coated 10 cm culture dish.
- Culture for o/n.
Day 2
- Collect supernate into 50 ml tube.
- OPTIONAL: Add growth medium to non-coated dish (fibroblast-rich) to keep remaining myoblast.
- Centrifuge at 2000 rpm at 10C for 5 min.
- Suspend in 10 ml growth medium with Fungizone.
- Seed on one collagen-coated 10 cm dish.
