Phosphate buffered saline without calcium and magnesium

To induce osteogenesis, primary dermal fibroblasts from a previously published subject 13 (link) ; individuals I4, I9, and I10; and 3 unaffected controls were plated in cell culture plates at a density of 5 × 10 4 cells/mL and treated for 28 days with osteogenic medium comprising DMEM-F12 supplemented with 10% FBS , 1% P/S (100 U/ml and 100 μg/mL, respectively), 50 μg/mL ascorbate-2-phosphate (Sigma Aldrich), 10 -8 M dexamethasone (Sigma Aldrich), and 10 -2 M β-glycerophosphate (Sigma Aldrich). The presence of calcium deposits in induced cultures was determined by Alizarin Red S (Sigma Aldrich) staining as follows: cells were fixed in 10% formalin (Sigma Aldrich) for 10 minutes, then washed in phosphate-buffered saline without calcium and magnesium (ThermoFisher Scientific), and incubated for 10 minutes at 37 ºC with filtered 2% Alizarin Red S in water. Cells were finally washed several times to remove the excess stain and observed using Axiocam 105 color (Zeiss) connected to Eclipse TE300 Microscope (Nikon). The intensity of the red color in the images was quantified using the ImageJ software (Supplemental Figure 2, Supplemental Methods). 22 (link)