Analysis of calcium absorption through cGM was performed in a NUNC® carrier plate transwell system (insert position: medium). 5 mg of cGM were added in the upper chamber to 24 h pre-seeded SaOS-2 cells (6•104 cells/Well) and gently shaken on orbital shaker (70–80 rpm) for 30 min at 37 °C and 5% CO2. Samples were incubated under static conditions for further 24 h and medium was changed to OM. Supernatants were collected in reaction tubes. Calcium absorption was measured by Calcium AS FS (Diasys, Holzheim, Germany) according to the manual. Deficits of calcium in supernatants were calculated as absorption by the cGM.
For another experiment, calcium concentration was increased up to 15% to compensate for absorbed calcium. To analyze mineralization of cells and microtissues, medium was removed and samples were carefully washed with warm PBS (37 °C). For cell lysis, 0.5 N HCl (Th. Geyer, Berlin, Germany) was added, and samples were incubated at room temperature (RT) for 24 h during shaking. Afterwards samples were transferred to reaction tubes, incubated in an ultrasonic bath for 2 h and shaken for further 12 h at RT. Samples were measured as described above and calcium content was calculated relative to DNA content per well.
For another experiment, calcium concentration was increased up to 15% to compensate for absorbed calcium. To analyze mineralization of cells and microtissues, medium was removed and samples were carefully washed with warm PBS (37 °C). For cell lysis, 0.5 N HCl (Th. Geyer, Berlin, Germany) was added, and samples were incubated at room temperature (RT) for 24 h during shaking. Afterwards samples were transferred to reaction tubes, incubated in an ultrasonic bath for 2 h and shaken for further 12 h at RT. Samples were measured as described above and calcium content was calculated relative to DNA content per well.