Skeletal Myogenesis in vitro

Materials and Reagents

1. Collagen type-I coated plates (sterilized by UV overnight after coating) (the protocol for coating plates is provided by Sigma-Aldrich in the associated product information)
   
2. Myogenic cells in sterile conditions (Refer to Chen et al., 2014 and Gharaibeh et al., 2008 for primary human and mouse cell isolation respectively)
   Note: For mouse cells, C2C12 cell line (ATCC, catalog number: CRL-1772) may be used as a positive control.
   
3. DMEM high-glucose (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 11995 )
   
4. Fetal bovine serum (FBS) (Invitrogen, catalog number: 10437-028 )
   Note: Currently, it is “Thermo Fisher Scientific, Gibco^TM, catalog number: 10437-028”.
   
5. Horse serum (HS) (Invitrogen, catalog number: 26050-088 )
   Note: Currently, it is “Thermo Fisher Scientific, Gibco^TM, catalog number: 26050-088”.
   
6. Chicken embryo extract (CEE) (Accurate Chemical & Scientific Corporation,  catalog number: MD-004-D )
   
7. Penicillin-Streptomycin (P/S) (Invitrogen, catalog number: 15140-122 )
   Note: Currently, it is “Thermo Fisher Scientific, Gibco^TM, catalog number: 15140-122”.
   
8. Monoclonal Anti-Desmin antibody produced in mouse (1:100) (Sigma-Aldrich, catalog number: D1033 ), Mouse anti-fast-MyHC (1:250) (Sigma-Aldrich, catalog number: M4276 ) or Monoclonal Anti-Myosin (Skeletal, Slow) antibody produced in mouse (1:250) (Sigma-Aldrich, catalog number: M8421 )
   
9. Normal donkey serum (Jackson Immuno Research, catalog number: 017-000-121 )
   
10. Alexa 594-conjugated anti-mouse IgG antibody (1:500) (Invitrogen, Molecular Probes, catalog number: A-21203 ) or Alexa 488-conjugated anti-mouse IgG antibody (1:500) (Invitrogen, Molecular Probes, catalog number: A-21202 )
    Note: Currently, it is “Thermo Fisher Scientific, Novex^TM, catalog numbers: A-21203 and A-21202”.
    
11. 4',6-diamidino-2-phenylindole (DAPI) (100 ng/ml, diluted with DPBS from the stock) (Sigma-Aldrich, catalog number: D9542 )
    
12. Mouse-on-mouse (M.O.M) Basic kit (staining kit) (Vector Labs, catalog number: BMK-2202 )
    
13. Collagen from calf skin (non-sterile) (Sigma-Aldrich, catalog number: C9791 )
    
14. Dulbecco’s DPBS without calcium and magnesium (DPBS) (Invitrogen, catalog number: 14190-250 )
    Note: Currently, it is “Thermo Fisher Scientific, Gibco^TM, catalog number: A-21203 and A-21202”.
    
15. Methanol (Sigma-Aldrich, catalog number: 322415 )
    
16. Acetone (Sigma-Aldrich, catalog number: 270725 )
    
17. Formalin (10%) (Sigma-Aldrich, catalog number: HT501128 )
    
18. Trypsin-EDTA (1x, diluted from the 10x stock with sterile DPBS, no phenol red) (Invitrogen, catalog number: 15400-054 )
    Note: Currently, it is “Thermo Fisher Scientific, Gibco^TM catalog number: 15400-054”.
    
19. Collagen type-I (Sigma-Aldrich, catalog number: C9791)
    
20. Muscle proliferation medium (see Recipes)
    
21. Muscle fusion medium (see Recipes)
    

Equipment

1. Cell culture incubator
2. Fluorescence microscope
   

Procedure