room temperature rt the monolayers were then washed with double distilled water ddh 2 o u00d7 3 incubated with 1 alizarin red stain for 10 min washed again with ddh 2 o u00d7 3 and air dried images were taken using an olympus bx51 microscope dp70 camera and dpcontroler software v1 1 1 65 for phosphate nodule staining cell monolayers were then washed with pbs u00d7 3 and incubated with 4 pfa for 10 min at rt cell monolayers were then washed with ddh 2 o u00d7 3 and incubated for 30 min in 1 silver nitrate under uv light the monolayers were then washed with ddh 2 o u00d7 3 incubated with 5 sodium thiosulfate for 2 min and washed again with ddh 2 o u00d7 3 and air dried images were taken using an olympus bx51 microscope dp70 camera and dpcontroler software v1 1 1 65 bioquant image analysis u00ae software bioquant image analysis corporation tn usa was used to quantify the average density of alizarin red s and von kossa stained wells briefly cultures were performed in triplicate 21 day cultures mml hs and fml hs treated cultures for each concentration were performed on the same plate to ensure the same light intensity and exposure within the samples images of the plates were taken using an epson perfection 1670 scanner using the image analysis software the digital images were loaded into the field of view and a consistent region of interest roi was placed over each well the average density of the.