Decorticated Bone Tissue Sclerostin and IL-1β Measurement

The level of sclerostin secreted by Ocy454 cells and the levels of IL-1β and sclerostin in rat bone tissues of transverse process were measured using a sclerostin ELISA kit (SU-B31033, Kenuodi, Quanzhou, China) and a IL-1β ELISA kit (SU-B30419, Kenuodi, Quanzhou, China) according to the manufacturer’s instructions. Transverse processes of at L4 and L5 in rats were decorticated using a drill. Decorticated bone tissues were harvested at day 1, 3, 7, 14 and 28. Transverse processes of L4 and L5 in unoperated rats were used as control (day 0). Equal weight of bone tissue was repeatedly crushed in 5 ml PBS and the supernatant was collected as previously reported [32 (link), 33 (link)]. The standard samples, experimental samples and antibody were added to a microplate. After three washes, the 3,3′,5,5′-tetramethylbenzidine colourimetric reagent and the stop solution were added to the wells in succession. The absorbance at 450 nm was measured using a microplate reader. The concentrations of IL-1β and sclerostin were calculated from standard curves.