Ultra deep freezer

At the end of the experiment with continuous supply of test substance for 84 days, blood from vena cava of each mouse was collected under inhalation anesthesia with 3% isoflurane for induction and 1.5–1.8% isoflurane for maintenance of anesthesia (in the mixture of 28.5% O₂ and 70% N₂O; Hana Pharm. Co., Hwasung, Korea) using rodent inhalation anesthesia apparatus (Surgivet, Waukesha, WI, USA) and rodent ventilator (Harvard Apparatus, Cambridge, UK). The blood samples were stored in NaF glucose vacuum tubes (Becton Dickinson, Franklin Lakes, NJ, USA), and plasma was separated and stored in an ultra-deep freezer (Sanyo, Tokyo, Japan) at −150 °C until further analysis. An automated blood analyzer (Fuji Medical System Co., Ltd., Tokyo, Japan) was used to measure blood glucose levels.

At sacrifice, whole blood (approximately 1 ml) was collected in a serum collection tube and the serum was separated by centrifugation at 15,000 rpm for 10 min under 4 °C. All collected samples were stored at − 150 °C using an Ultra-deep freezer (Sanyo, Tokyo, Japan) until further analysis. Mouse Osteocalcin ELIZA Kit and Mouse bALP ELIZA Kit, purchased from MyBioSource (CA, USA), were used for measuring the serum osteocalcin and bALP levels, respectively.

Serum biochemical analyses were performed as demonstrated in our previous study [43 (link)]. Approximately 200 μL of whole blood sample was drawn from the posterior vena cava using a 26-gauge needle attached syringe under 2–3% isoflurane (Hana Pharm., Hwaseong, Korea) inhalation anesthesia. Blood samples were collected using a separation tube and centrifuged at 1000× g for 10 min. Separated serum samples were stored at −150 °C in an ultra-deep freezer (Sanyo, Tokyo, Japan) till further analysis. Serum creatinine, creatine kinase (CK) and lactate dehydrogenase (LDH) levels were measured using auto analyzer (Fuji Medical System Co., Ltd., Tokyo, Japan). Animals were sacrificed by cervical dislocation.