Ac 06f1

Serum concentrations were measured using commercially available ELISAs for PINP (SEA957Mu, Cloud Clone Corp.), CTX (AC-06F1, Immunodiagnostic Systems), RANKL (MTR00, R&D Systems) and OPG (#MOP00, R&D Systems).

All quantifications were made at the Department of Clinical Pathology of São João Hospital Centre EPE, Porto, Portugal. Circulating concentrations of N-terminal propeptide of type I procollagen (PINP; AC-33F1 from Immunodiagnostic Systems Limited, Tyne & Wear, United Kingdom), osteopontin (OPN; JP27360 from Immuno-Biological Laboratories, Co. Limited, Gunma, Japan), osteocalcin (OTN; AC-12F1 from Immunodiagnostic Systems Limited, Tyne & Wear, United Kingdom), and degradation products from C-terminal telopeptides of type I collagen (RatLapsTM, CTX-1; AC-06F1 from Immunodiagnostic Systems Limited, Tyne & Wear, United Kingdom) were evaluated by using enzyme immunoassay/enzyme-linked immunosorbent assay (EIA/ELISA), according to the instructions provided by the suppliers. Circulating concentrations of receptor activator of nuclear factor-kappaB ligand (RANKL; RRNKLMAG-31K-01, Single Plex) and osteoprotegerin (OPG; RBN1MAG-31K, two plex) were determined by a plex bead assay performed according to protocols (MILLIPLEX^® MAP kits) of Millipore Corporation (Billerica, MA, USA). Quantifications were performed using a Luminex 200 analyzer (Luminex Corporation, Austin, TX, USA).

Serum alkaline phosphatase (ALP), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatinine kinase (CK), blood urea nitrogen (BUN) levels, serum creatinine (CRE) levels, and urinary creatinine (U-CRE) levels were measured by Nagahama Life Science Laboratory, Oriental Yeast Co., Ltd. (Shiga, Japan). Plasma corticosterone levels on the second day and weeks two and four were determined using commercially available EIA kits (YK2 40, Yanaihara Ins. Inc., Shizuoka, Japan). Serum levels of Gla-osteocalcin (Gla), a bone formation marker, were determined using a mouse Gla-OC competitive enzyme immunoassay (EIA) kit (MK111, TAKARA Biomedicals, Kyoto, Japan). Bone resorption markers, including serum tartrate-resistant acid phosphatase-5b (TRAP-5b) and urinary C-terminal telopeptides of type I collagen (CTX), were evaluated using a mouse TRAP assay kit (SB-TR103, Immunodiagnostic Systems, Fountain Hills, AZ, USA) and a commercial EIA kit (AC-06F1, Immunodiagnostic Systems, Fountain Hills, AZ, USA), respectively. Urinary CTX levels were corrected by urinary CRE concentrations.

Serum was collected from mice by cardiac puncture under terminal anaesthetic. Briefly, whole blood was left at room temperature for 30 min prior to centrifugation for 20 min at 12,000 rpm. Serum was aspirated and stored at − 80 °C prior to analysis. Unbound, serum-free corticosterone levels were measured using a commercially available sandwich ELISA designed to specifically detect active (but not inactive 11DHC) steroid (cat no: KGE009, R&D systems, Abingdon, UK). Serum was analysed in accordance with the manufacturer’s instructions and data expressed as nanogrammes per millilitre (ng/ml). Serum P1NP was determined using a commercially available sandwich ELISA (cat no: AC-33F1, Immunodiagnostic Systems, Tyne & Wear, UK) in accordance with the manufacturer’s instructions and data expressed as ng/ml. Serum CTX-1 was determined using a commercially available sandwich ELISA (cat no: AC-06F1, Immunodiagnostic Systems, Tyne & Wear, UK) in accordance with the manufacturer’s instructions and data expressed as units per microlitre.

The bone resorption biomarker C-telopeptide of type 1 collagen (CTX) (Immunodiagnostic Systems, Cat#AC-06F1) and the bone formation marker propeptide of type 1 collagen (P1NP) (Immunodiagnosticsystems; Cat#AC-33F1) were analyzed in serum from mice or in conditioned media from bones cultured ex vivo, as previously described (9 (link), 14 (link)).

Blood was collected in heparin‐coated tubes and centrifuged (2000g; 10 minutes; RT) to collect the supernatant (plasma). N‐terminal propeptide of type I procollagen (PINP) (cat. AC‐33F1; Immunodiagnostic Systems, Boldon, UK) and C‐terminal telopeptides of type I collagen (CTX‐I) ELISAs (AC‐06F1; Immunodiagnostic Systems) were performed according to the manufacturer's instructions.