Ratlaps ctx 1 eia

The serum level of total Ocn was examined using the Ocn EIA kit (BTI Biomedical Technologies, Inc., Stoughton. MA), that of carboxyleted Ocn by the Mouse Gla-Osteocalcin High Sensitive EIA Kit (Takara Bio Inc., Shiga Japan), that of uncarboxyleted Ocn by the Mouse Glu-Osteocalcin High Sensitive EIA Kit (Takara Bio Inc.), that of P1NP by Rat/Mouse P1NP ELISA (Immunodiagnostic Systems, Boldon, UK), that of TRAP5b by the Mouse TRAP Assay (Immunodiagnostic Systems), and that of CTX1 by RatLaps (CTX-I) EIA (Immunodiagnostic Systems). Serum testosterone levels were assessed using Testosterone Rat/Mouse ELISA (Demeditec Diagnostics GmbH, Kiel, Germany).

To determine the concentrations of bone turnover markers (procollagen type I N-terminal propeptide (PINP); OPG; C-terminal telopeptide (CTX); RANKL), blood was harvested at an age of 12 or 36 weeks and 8 weeks after OVX or sham operation in microvettes (Sarstedt AG & Co., Nümbrecht, Germany), centrifuged at 13,000 × g for 7 min for serum collection and stored at −80°C until further use. Bone turnover markers were determined using RatLaps™ CTX-I EIA, Rat/Mouse PINP EIA (both from Immunodiagnostic Systems GmbH, Frankfurt am Main, Germany), mouse RANKL ELISA Kit (TNFSF11), and mouse Osteoprotegerin ELISA Kit (TNFRSF11B) (both from Abcam, Cambridge, United Kingdom) according to manufacturers’ protocols. Using a customized mouse Multiplex Cytokine Kit (ProcartaPlex, eBiscience, Frankfurt, Germany), the CXCL-1, CXCL-10, IL-6, monocyte chemoattractant protein-1 (MCP-1), macrophage colony-stimulating factor (M-CSF), and vascular endothelial growth factor (VEGF) concentrations were determined in the OVX and Sham mice.

Female mice were harvested at 5 weeks of age. Blood samples were collected immediately after euthanization by opening the peritoneal cavity and revealing the posterior vena cava for puncture using a 25G needle and a 1‐mL syringe. The blood samples were collected into Micro 1.1‐mL Z‐Gel tubes (Sarstedt, Numbrecht, Germany) and centrifuged at 10,000g for 10 minutes at room temperature to separate the serum. Serum levels for the N‐terminal propeptide of type I procollagen (PINP) and cross‐linked C‐terminal telopeptide of type I collagen (CTX‐I) were measured using commercial enzyme immunoassay (EIA) kits (Rat/Mouse PINP EIA and RatLaps [CTX‐I] EIA, respectively) (Immunodiagnostic Systems, Boldon, UK), according to the manufacturer's instructions. The mice were not fasted before CTX‐I measurements.

Analyses of alkaline phosphatase (AP), calcium (Ca), magnesium (Mg), and phosphor (P) were conducted at the Department of Clinical Chemistry, University Medical Center, Goettingen using commercial tests (Architect, Abbott, Wiesbaden, Germany) and an automated chemistry analyzer (Architect c16000 Analyzer, Abbott). Osteocalcin (OC) and the cross-linked C-telopeptide of type-I collagen (CTX-I) were assessed using an enzyme-linked immunosorbent assay (EIA), rat-MID™ Osteocalcin EIA, and RatLaps (CTX-I) EIA, respectively (Immunodiagnostic Systems GmbH, Frankfurt am Main, Germany). Follicle stimulating hormone (FSH) and luteinizing hormone (LH) were measured by EIA kit for rats (Cloud-Clone Corp., Katy, Texas, USA).

Sera were collected from mice through retro-orbital bleeding after 6 hours of fasting. CTX-I and P1NP assays were performed with the RatLaps^™ (CTX-I) EIA and Rat/Mouse P1NP EIA Kit (Immunodiagnostic Systems, Ltd.), respectively. Insulin and Igf1 assays were performed with mouse insulin ELISA kit (Ultra Sensitive Mouse Insulin ELISA, Cat#90080, Crystal Chem) and mouse Igf1 ELISA kit (Mouse IGF-1 ELISA Kit, Cat#80574, Crystal Chem). HOMA-IR was calculated as follows: HOMA IR = fasting insulin (mU/L) * fasting glucose (mg/dL)/405. Fasting glucose levels were measured with a glucometer in blood collected from a tail-vein cut.