Ratlaps elisa

Serum was collected from mice after 6 hours of fasting for CTX-I and PINP assays. The assays were performed with the RatLaps ELISA or Rat/Mouse PINP EIA Kit (both from Immunodiagnostic Systems, Ltd.).

Ten-weeks-old female C57BL6 mice were ovariectomized under complete anesthesia. Two weeks after the operation, the mice were injected s.c. twice a day for 3 d with PBS or 10 mg/kg of M-CSF_RGD variants 4.22 or 5.6. Three h after the last injection, the mice were anesthetized with isoflurane, and their blood was collected through a cardiac puncture. Then, blood samples were centrifuged at 4,000 × g for 10 min, and the serum samples were transferred to fresh tubes. To determine the serum CTX-I levels, a RatLaps ELISA (Immunodiagnostic systems, UK) was used according to manufacturer’s protocol. Hemolytic samples were not used.

For serum cross-linked C-telopeptide (CTX-I) and amino-terminal propeptide of type I procollagen (P1NP) assays, serum was collected from mice after 6 h of fasting. Assays were performed with RatLaps ELISA or Rat/Mouse P1NP EIA kit (both from Immunodiagnostic Systems, Ltd.).

For serum cross-linked CTX-I and amino-terminal P1NP assays, serum was collected from mice after 6 h of fasting. Assays were performed with the RatLaps ELISA or Rat/Mouse P1NP EIA Kit (both from Immunodiagnostic Systems, Ltd.).

Blood was obtained from anesthetized mice by cardiac puncture at 6 weeks of age, and sera were stored in aliquots at -80°C. All ELISAs were performed as instructed by the manufacturer. C-terminal telopeptide (CTX) measurements were performed using the RatLaps ELISA (Immunodiagnostic Systems, Gaithersburg, MD; #AC-06F1). TRAcP 5b protein measurement was performed using Mouse TRAP ELISA (Immunodiagnostic Systems; #SB-TR103). Osteocalcin measurement was performed using the Mouse Osteocalcin EIA kit (Biomedical Technologies, Ward Hill, MA # BT-470). Assays were run in triplicate on between 3 and 9 individual animals per genotype.