DMEM/F-12

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	Three thousand normal human osteoblasts were seeded in 24-well plates (Fisher Scientific) containing 1 mL media with different MgCl2 concentrations 0 mM, 0.5 mM, 1.0 mM, 2.0 mM, 4.0 mM, 8.0 mM and 16.0 mM MgCl2 concentrations (Fisher Scientific). Each condition was triplicated and growth medium was changed every three days. The culture plates were incubated under 37̊C and 5% CO2 for following times: 16 hours, 7 days, 10 days, 14 days and 21 days. Cell attachment was quantified at 16 hours, through direct cell counts and normalized to the cell seeding density. Cell proliferation was determined at time points of 7, 10, 14 and 21 days.

Protocol tips

Three thousand normal human osteoblasts were seeded in 24-well plates (Fisher Scientific) containing 1 mL media with different MgCl2 concentrations 0 mM, 0.5 mM, 1.0 mM, 2.0 mM, 4.0 mM, 8.0 mM and 16.0 mM MgCl2 concentrations (Fisher Scientific). Each condition was triplicated and growth medium was changed every three days. The culture plates were incubated under 37̊C and 5% CO2 for following times: 16 hours, 7 days, 10 days, 14 days and 21 days. Cell attachment was quantified at 16 hours, through direct cell counts and normalized to the cell seeding density. Cell proliferation was determined at time points of 7, 10, 14 and 21 days.

Publication protocol

Three thousand normal human osteoblasts were seeded in 24-well plates (Fisher Scientific) containing 1 mL media with different MgCl2 concentrations 0 mM, 0.5 mM, 1.0 mM, 2.0 mM, 4.0 mM, 8.0 mM and 16.0 mM MgCl2 concentrations (Fisher Scientific). Each condition was triplicated and growth medium was changed every three days. The culture plates were incubated under 37̊C and 5% CO2 for following times: 16 hours, 7 days, 10 days, 14 days and 21 days. Cell attachment was quantified at 16 hours, through direct cell counts and normalized to the cell seeding density. Cell proliferation was determined at time points of 7, 10, 14 and 21 days.

At each predetermined time point, growth medium was removed and cells were fixed with 10% neutral buffered formalin (Sigma) for one hour at room temperature. Fixation solution was then discarded and plates were washed twice with PBS and stained with 1 mL 0.2% crystal violet stain (Sigma Aldrich) for one hour at room temperature. Unbound stain was removed by rinsing the plates thoroughly with deionized water until clear. Then optical density value was measured by a microplate reader (TECAN, Infinite 200 Pro) at a wavelength of 590 nm. Absorbance of crystal violet is directly proportional to cell numbers. Three measurements were performed and mean cell numbers were calculated by using a standard curve.

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