Crosslaps for culture elisa

In order to evaluate bone resorption, formation of resorption pits on bone slices was analyzed by Coomassie brilliant blue staining. After 6 days of culture, bone slices were washed with water and the cells were removed by sonication for 30 min in 10% ammonia (Merck, Darmstadt, Germany) on ice. To reduce staining background the bone surface was incubated in saturated alum for 10 min and subsequently washed with water. Resorption pits were stained with Coomassie brilliant blue (Pharmacia, Uppsala, Sweden) and visualized by light microscopy (Leica DFC320). In addition, CTX concentrations of the culture supernatants were determined using CrossLaps® for culture ELISA (Immunodiagnostic Systems Limited, Frankfurt am Main, Germany). Culture supernatants were collected after 6 days and CTX assay was conducted following the manufacturer's instructions.

Conditioned media was collected after the first 48 h and centrifuged at 1500 RPM for 5 min, and the supernatant was collected and stored at − 80 °C. Conditioned media was warmed to room temperature, and CrossLaps® for Culture ELISA (Immunodiagnostic Systems, Inc., Fountain Hills, AZ) was used to determine the degradation marker, C-terminal telopeptide of type I collagen, from the breakdown of the contracted cell-seeded gel according to the manufacturer’s instructions. Briefly, preparation a two-fold dilution row of the standards, pre-dilution of test specimens with standard diluent and preparation of antibody solution were performed before the test. Then each of standards, control and samples were pipetted into appropriate wells to mix with antibody solution and incubated at room temperature for 2 h. Wash with diluted washing buffer for 5 washing cycles followed by adding substrate solution into each well and incubating for 15 min at room temperature in the dark on the mixing apparatus. Finally, add stopping solution into each well followed by measuring the absorbance at 450 nm in an absorbance microplate reader.