Free calcium concentration from medium after the 7-day incubation period of MG-63 cells was performed using a Calcium Calorimetric Assay Kit (Sigma-Aldrich, Gillingham, UK). Calcium ion concentration was measured by measuring the chromogenic complex formed between o-cresolphthalein and calcium ion. The chromogenic complex was determined spectrophotometrically by measuring the optical density at 575 nm. The optical density was proportional to the concentration of calcium ions present in the samples.
The assay was performed as per manufacturer’s instructions. Briefly, 50 μL of samples or calcium standards were added to a 96-well plate. 90 μL of chromogenic reagent and 60 μL of calcium assay buffer was added to wells containing controls, samples, or standards. The assay plate was incubated for 10 min at room temperature and absorbance was measured at 575 nm using microplate reader.
The assay was performed as per manufacturer’s instructions. Briefly, 50 μL of samples or calcium standards were added to a 96-well plate. 90 μL of chromogenic reagent and 60 μL of calcium assay buffer was added to wells containing controls, samples, or standards. The assay plate was incubated for 10 min at room temperature and absorbance was measured at 575 nm using microplate reader.