Tracp 5b

At the end of the in vivo experiments (2 weeks of continuous hPTH infusion), blood samples were collected by cardiac puncture from the mice. Blood was allowed to clot at room temperature and sera collected after centrifugation of blood samples at 5000 rpm for 10 min. Serum MCP-1 concentrations were measured by using the Quantikine ELISA (R&D Systems). Serum C-terminal crosslinking telopeptide of type I collagen (CTX, Immunodiagnostic Systems Inc.), P1NP (MyBiosource.com) and TRACP-5b (Immunodiagnostic Systems Inc.) were measured by rodent-specific ELISAs. Serum total calcium was measured by calcium colorimetric assay kits (BioVision) in accordance with the manufacturer’s instructions.

Tartrate-resistant acid phosphatase 5b (TRACP-5b, Immunodiagnostic Systems Limited, Bolden, UK) and osteocalcin (Elabscience, Wuhan, China) levels in mouse serum were measured by specific ELISA Kits. Experiment was performed in duplicate and the absorbance was measured using MULTISKAN JX (Thermo Fisher Scientific). Values were analyzed using Ascent Software for Multiskan Ascent (Thermo Fisher Scientific).

Serum concentrations of albumin, urea nitrogen, sodium, calcium, and phosphate were measured with an automated analyzer (Hitachi, Tokyo, Japan). Serum levels of intact parathyroid hormone (PTH) (Immutopics International, San Clemente, CA), osteocalcin (Biomedical Technologies, Stoughton, MA), and tartrate‐resistant acid phosphatase‐5b (TRACP‐5b) (Immunodiagnostic Systems, Gaithersburg, MD) were determined using commercially available mouse ELISA kits. The kits were used according to the manufacturer's instructions, and their qualities were within analytical levels.

Cardiac blood collected at time of euthanasia was used to measure serum blood urea nitrogen (BUN) via colorimetric assay as an assessment of presence/absence of kidney disease (BioAssay Systems). Serum 1–84 PTH was measured via ELISA (Immnotopics Quidel) with an intra-assay coefficient of variation of 2.4%–5.6% and an inter-assay coefficient of variation of 5.5%. Serum tartrate-resistant acid phosphatase 5b (TRAcP 5b) was measured as an assessment of systemic osteoclast numbers (Immunodiagnostic Systems). The intra-assay coefficient of variation for serum TRAcP 5b is 3.3%–6.9% and the inter-assay coefficient of variation is 5.2%–7.2%. All serum assays were run in duplicate with serum samples that had not been through any freeze/thaw cycles and followed all the procedures of the manufacturer.