Materials and Reagents

1. Multipotent stromal cells (MSC) isolated from mouse bone marrow [see protocol “Isolation of Multipotent Stromal Cells from Mouse Bone Marrow” (Tormo et al., 2014)]
   
2. Dulbecco’s Modified Eagle’s Medium High glucose with stable L-glutamine (DMEM) (Wisent, catalog number: 319-015-CL )
   
3. Penicillin/Streptomycin solution (Wisent, catalog number: 450-201-EL )
4. Fetal Bovine Serum (FBS) (Life Technologies, Gibco^®, catalog number: 12483 )
   
5. 0.53 mM 0.05% Trypsin/EDTA (Wisent, catalog number: 325-042-EL )
   
6. PBS without Ca²⁺ and Mg²⁺ (Wisent, catalog number: 311-01-CL )
   
7. HEPES (Wisent, catalog number: 330-050-EL )
   
8. 10 nM Dexamethasone (Sigma-Aldrich, catalog number: D4902 )
   
9. 50 μM Ascorbic acid-2 phosphate (vitamin C) (Sigma-Aldrich, catalog number: A4403 )
   
10. 50 nM Cholecalciferol (Vitamin D3) (Sigma-Aldrich, catalog number: C9756 )
    
11. 10 mM B-glycerophosphate (Sigma-Aldrich, catalog number: G9891 )
    
12. 100x Protease and phosphatase inhibitors (Thermo Fisher Scientific, Pierce, catalog number: PI78447 )
    
13. IL-27 (1 μg) (R&D Systems, catalog number: 2799-ML-010/CF ) or any recombinant protein to be tested
    
14. Bicinchoninic acid assay (BCA) (Thermo Fisher Scientific, Pierce, catalog number: PI23225 )
    
15. Distilled water
    
16. Ethanol
    
17. Anti-IL-27 p28 biotinylated antibody (R&D Systems, catalog number: BAF1834 ) or biotinylated antibody specific for the protein to be tested
    
18. Streptavidin-HRP (Thermo Fisher Scientific, Pierce, catalog number: PI21130 )
    
19. SDS-PAGE loading buffer without bromophenol blue or DTT
    
20. Urea buffer (Fisher Scientific, catalog number: BP169-10 )
    
21. 1 M Sodium phosphate monobasic (Fisher Scientific, catalog number: BP330-1 )
    
22. 1 M Sodium phosphate dibasic (Fisher Scientific, catalog number: BP332-1 )
    
23. TBS-Tween
    
24. 3% BSA
    
25. Dexamethasone solution (see Recipes)
    
26. 10x Glycerophosphate solution (see Recipes)
    
27. 1,000x Vitamin C solution (see Recipes)
    
28. Osteogenic differentiation medium (50 ml) (see Recipes)
    
29. Alizarin red S (Sigma-Aldrich, catalog number: A5533 ) (see Recipes)
    
30. Urea/Phosphate lysis buffer (see Recipes)
    

Equipment

1. 15 ml tube
   
2. Centrifuge for cell culture
   
3. 6 well tissue culture plates (BD Biosciences, catalog number: DL-353046 )
   
4. 96 well tissue culture plates with flat bottoms (BD Biosciences, catalog number: DL-353072 )
   
5. Inverted phase-contrast microscope
   
6. 37 °C, 5% CO₂ cell culture incubator
   
7. Cell scrapers (Corning, catalog number: 3010 )
   
8. Microplate reader for BCA (562 nm)
   
9. SDS-PAGE Equipment
   
10. Western blot Equipment
    

Procedure