Ab100749

The ratio of RANKL to osteoprotegerin (OPG) mRNA expression was compared for pre-osteoblasts and osteoblasts using quantitative PCR on synthesized cDNA (Section 4.3), Applied Biosystems TaqMan Gene Expression Master mix, and targeted TaqMan gene expression assays for RANKL (Mm00441906_m1) and OPG (Mm00435454_m1).
Protein was isolated from pre-osteoblasts, osteoblasts, pre-osteoclasts, and osteoclasts treated for 7 days with control or sertraline low (34.2 ng/ml) or high (342 ng/ml) dose supplemented media. Cells were seeded in triplicate at a density of 300,000 cells per well and supernatant was collected before cells were washed with cold Phosphate Buffered Saline (PBS, HyClone, Fisher Scientific, SH30256.01) and protein was extracted using radioimmunoprecipitation buffer (RIPA, G Biosciences, St. Louis, MO, 786–490) for 30 mins at 4°C with agitation. Supernatant and cell lysate was collected and used for RANKL (AbCam, Cambridge, MA, Ab100749) and OPG (AbCam, Ab100733) ELISA according to manufacturer protocol. The ratio of RANKL and OPG was calculated for each cell type for each treatment in both cell lysates and supernatant. Each assay was repeated three times.

This assay was used to determine the amount of RANKL and OPG in the bone marrow plasma of the different experimental mice. BM plasma-derived RANKL and OPG was measured using commercial mouse RANKL (ab100749) and OPG (ab100733) sandwich ELISA kits from Abcam, as per the manufacturer’s instructions. To determine JNK protein phosphorylation and the pathway activation in BM cells in the experimental mice, we used JNK (Thr183/Tyr185) In-Cell ELISA kit (ab126424) from Abcam, as per the manufacturer’s instructions.