Materials and Reagents

1. Cardiac ventricular fibroblasts isolated from neonatal Sprague-Dawley rats by enzymatic dissociation (Lee et al., 2013)
   Note: Our protocol can be used with various cell types such as osteoblasts and vascular smooth muscle cells.
   
2. Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose and 4 mM L-glutamine (Hyclone, catalog number: SH 30243.01 )
   
3. Fetal Bovine Serum (FBS) (Hyclone, catalog number: SH 30919.03 )
   
4. Inorganic phosphate (Pi) (pH 7.4)
   
5. DPBS without Ca²⁺ and Mg²⁺ (Hyclone, catalog number: SH 30028.02 )
   
6. Trypsin/EDTA (Hyclone, catalog number: SH 30042.01 )
   
7. 70% ethanol
   
8. Distilled deionized water (DDW)
   
9. 0.6 N HCl solution
   
10. Lysis solution
    
11. 0.1 N NaOH
    
12. 0.1% sodium dodecyl sulphate (SDS)
    
13. QuantiChrome Calcium Assay Kit (BioAssay Systems, catalog number: DICA-500 )
    
14. Working reagent (refer to QuantiChrome Calcium Assay Kit manual for detail) (BioAssay Systems, catalog number: DICA-500) (see Reference 2)
    
15. Bio-Rad DC protein assay kit (Bio-Rad Laboratories, catalog number: 500-0016 )
    
16. Diluted protein standards (e.g., 0, 2, 4, 6, 8, 12, 16 and 20 mg/dl)
    
17. 100 ng/ml receptor activator of NF-kB ligand (RANKL) (Sigma-Aldrich, catalog number: R0525 )
    
18. 5% Aqueous silver nitrate solution (Sigma-Aldrich, catalog number: S7279 ) (see Recipes)
    
19. 5% sodium thiosulfate (Sigma-Aldrich, catalog number: S7026 ) (see Recipes)
    
20. 2% alizarin red S solution (Sigma-Aldrich, catalog number: A5533 ) (see Recipes)
    
21. 1 M NaH₂PO₄ (pH 7.4) (Sigma-Aldrich, catalog number: S6566 ) (see Recipes)
    

Equipment

1. 48-well plate
   
2. 96-well plate
   
3. Spectrophotometer or 96-well reader
   
4. 37 °C, 5% CO₂ cell culture incubator
   
5. Inverted microscope
   
6. Aspirator
   
7. UV-visualizer
   
8. High-watt lamp (60-100 watt)
   

Procedure