Quantikine elisa human osteocalcin immunoassay

For Osteocalcin secretion measurements, the cell-seeded structures were prepared using Quantikine^®ELISA Human Osteocalcin Immunoassay (Catalog Number DSTCN0 (R&D SYSTEMS, Minneapolis, MN, USA)), according to the producer’s specifications. The standard curve for Osteocalcin calibration was obtained using standard osteocalcin solution in the kit. 50 µL from the supernatant from each sample was added to a 96-well plate, together with 100 µL of Assay Diluent. The samples were incubated while shaking during two hours and washed three times using the washing buffer. Next, 200 µL from the conjugate were added in each well. After two hours of shaking at room temperature, the samples were washed for four times with the washing buffer. Next, 200 µL from the substrate solution was added to each well and incubated during 30 min in the dark. The reaction was finished using 50 µL of the Stop solution. Osteocalcin secretion was measured through the absorbance at wavelength of 450 nm with a correction at 570 nm, using a Mitras LB 940 (Berthold Technologies) spectrophotometer.

In order to measure the Human Osteocalcin protein, the cells were seeded similarly as for SE; the samples were prepared using Quantikine^®ELISA Human Osteocalcin Immunoassay (Catalog Number DSTCN0 (R&D SYSTEMS, Minneapolis, MN, USA)), according to the producer’s specifications. The standard Osteocalcin solution in the kit was used in order to obtain a standard curve for Osteocalcin calibration. A total of 50 µL from the supernatant of each sample was added to a 96-well plate, together with 100 µL of Assay Diluent. The samples were incubated while shaking during 2 h; after this time, they were washed 3 times using the washing buffer; 200 µL from the conjugate were added in each well. After another 2 h of shaking at room temperature, the samples were washed 4 times using the washing buffer; 200 µL from the Substrate solution was added to each well and then allowed to incubate for 30 min, in the dark. At the end of this period, the reaction was finished using 50 µL of the Stop solution. The osteocalcin protein secretion was measured spectrophotometrically at 450 nm with a correction at 570 nm, using the Mitras LB 940 (Berthold Technologies).