分子細胞機能学研究室

ホーム研究実験業績メンバーリンク

Preparation of primary myoblast by MACS

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)
• Matrigel (-20C)
• MACS buf (4C)
• MACS column (big and small)
• 12 ml syringe with 18G needle
• 70 um Cell Strainer (Fisher Scientific, 22-363-548)
• @CD31-PE (eBioscience 12-0311, 4C)
• @CD45-PE (eBioscience 12-0451, 4C)
• @Sca1-PE (eBioscience 12-5981, 4C)
• @Integrin α7 (MBL K0046-3, -20C)
• @PE-MicroBeads (Miltenyi Biotec 130-048-801, 4C)
• @mIgG-MicroBeads (Miltenyi Biotec 130-048-401, 4C)

Dissection and dissociation of muscle

1. Euthanize mouse and peal skin.
2. Dissect pectoral, brachial, leg, and abdominal muscle.
3. Wash muscle by PBS on 10 cm petri dish.
4. Eliminate fat, tendon, and vessel.
5. Mince muscle in 5 ml collagenase II sol on 6 cm petri dish.
6. Incubate at 37C for 1 h.
7. Suspend by 12 ml syringe with 18G needle.
8. Incubate at 37C for 15 min.
9. Put into 25 ml DMEM in 50 ml tube.
10. Filtrate by 70 um Cell Strainer into four 50 ml tube with diluting by PBS.
11. Keep tube on ice in following procedure.

Collecting CD31(-)/CD45(-)/Sca1(-) cell

1. Centrifuge at 2000 rpm at 10C for 5 min.
2. Remove supernate with leaving 1 ml medium.
3. Suspend and move to 1.5 ml tube.
4. Centrifuge at 2000 rpm at 4C for 5 min.
5. Remove supernate.
6. Suspend together in 400 ul DMEM.
7. Add 1/200 @CD31-PE, @CD45-PE, @Sca1-PE, and @integrin α7.
8. Incubate on ice for 1 h.
9. Wash by 1 ml DMEM twice with centrifuging at 2000 rpm at 4C for 3 min.
10. Suspend in 200 ul DMEM.
11. Add 1/20 @PE-beads.
12. Incubate on ice for 30 min.
13. Wash by 1 ml MACS buf twice with centrifuging at 2000 rpm at 4C for 3 min.
14. Suspend in 1 ml MACS buf.
15. Wash MACS column by 2 ml MACS buf.
16. Add cell suspension to column with collecting flow-through.
17. Wash column by 2 ml MACS buf with collecting flow-through.

Collecting integrin α7(+) myoblast

1. Centrifuge at 2000 rpm at 4C for 5 min.
2. Suspend in 200 ul MACS buf.
3. Add 1/20 @mIgG-beads.
4. Incubate on ice for 30 min.
5. Prepare matrigel-coated dish.
6. Wash by 1 ml MACS buf twice with centrifuging at 2000 rpm at 4C for 3 min.
7. Suspend in 500 ul MACS buf.
8. Wash MACS column by 1.5 ml MACS buf.
9. Add cell suspension to column.
10. Wash column by 1.5 ml MACS buf.
11. Leave column from magnet.
12. Add 1 ml MACS buf.
13. Collect myoblast in column into 1.5 ml tube by syringe.
14. Count myoblast.
15. Centrifuge at 2000 rpm at 4C for 3 min.
16. Suspend in growth medium.
17. Seed myoblast on matrigel-coated dish.

Matrigel-coated dish

1. Dissolve matrigel on ice.
2. Coat 6 cm culture dish with matrigel on ice.
3. Incubate at 37C for 30 min.
4. Dry up.

Reference