- Polystyrene round bottom FACS tubes.
- 1PBS: 138 mM NaCl, 2.7 mM KCl, 1.5 mM KH 2 PO 4 ,
8mMNa 2 HPO 4 ·7H 2 O, pH 7.4. - Digestion buffer: DMEM/F-12, 10 mg/ml Collagenase B
(Roche, #11 088 831 001), 2.4 U/ml Dispase (Roche, #04
942 078 001). Sterilize by filtration. - Heat-inactivated fetal bovine serum (FBS).
- FACS buffer: 1PBS, 2% (v/v) FBS.
- Ammonium-chloride-potassium (ACK) buffer: 8 mg/l Nh 4 Cl,
1 mg/l KHCO 3 , 3.7 mg/l EDTA.Na 2 ·2H 2 O (Lonza, #10-
548E). - Antibody mix: FACS buffer, 5μg/ml anti α7integrin-647
(AB lab, #AB0000538), 2.5 μg/ml anti CD34-FITC
(ebioscience, #11-0341-82), 0.2 μg/ml anti CD45-PE
(ebioscience, #12-0451-82), 0.2 μg/ml anti CD31-PE
(ebioscience, #12-0311-82), 0.2 μg/ml anti Sca1-PE
(ebioscience, #12-5981-82). - 4^0 ,6-diamidino-2-phe ́nylindole (DAPI): 1 mg/ml stock
solution. - Cell sorting machine (e.g., Aria I).
2.2 Murine MuSC
Culture
- 0.22μm sterile bottle-top filter.
- 1PBS: 138 mM NaCl, 2.7 mM KCl, 1.5 mM KH 2 PO 4 ,
8mMNa 2 HPO 4 ·7H 2 O, pH 7.4. - Trypsin EDTA 0.25%: 0.25% (w/v) trypsin, 0.02% (w/v)
EDTA. - Coating solution: 10% (v/v) Matrigel in DMEM/F-12. Keep
the solution in ice until use (seeNote 1). - MuSC medium: DMEM/F-12, 20% (v/v) FBS, 100 U/ml
penicillin/streptomycin. Filter sterilizes using 0.22 μm
filter unit. - MuSC growth medium: MuSC medium, 2% (v/v) Ultroser™
G serum substitute (seeNote 2).
2.3 AMPK Activation/
Inhibition During
MuSC Differentiation
- MuSC differentiation medium: DMEM/F-12, 2% (v/v) horse
serum, 100 U/ml penicillin/streptomycin. - 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside
(AICAR): 75 mM stock solution in DMSO. - 991 compound: 10 mM stock solution in DMSO.
- 2 Deoxy-D-glucose (2-DG).
- STO-609: 10 mM stock solution in DMSO.
Muscle Stem Cells Fate 541