Mouse igf 1

The levels of mouse IGF-1 (R&D), VEGF (R&D), HGF (R&D), follistatin (Aviva Biosystems) and TGFß1 (R&D) were measured using ELISA, following the manufacturer’s instructions. Briefly, ELISA plates were incubated with standards, conditioned media from PICs or non-conditioned media (control) in triplicate for 2 h at room temperature, wells were rinsed with wash buffer five times and incubated with conjugate antibody or biotinylated antibody in the case of follistatin for 2 h at room temperature. After the second wash, follistatin plates only were incubated with 1x Avidin-HRP conjugate for 1 h at 37 °C before all plates were incubated with substrate solution for 20–30 min at room temperature or 37 °C in the case of follistatin. The addition of stop solution concluded the reaction and a microplate reader (Dynex Technologies, Chantilly, USA) was used to detect the signals at 450 nm with correction at 570 nm.

Terminal bleeds from mice were incubated at room temperature for 1 hour in serum separator tubes (Sarstedt, Nümbrecht, Germany) and then spun at 10,000 × rpm for 5 minutes. Serum was collected and stored at −80°C until analysis. Mouse leptin, mouse growth hormone, human NAG-1, mouse IGF-1 (R&D, Minneapolis, MN) and mouse insulin (Alpco, Salem, NH) ELISA kits were used according to manufacturer's instructions.

Mouse Wisp1 levels were determined using a Mouse/Rat Wisp-1/CCN4 Immunoassay (R&D Systems) that detects both natural and full-length recombinant Wisp1 protein. ELISAS to determine serum levels of mouse osteopontin, mouse osteoprogeterin and mouse Igf-1 were purchased from R&D Systems (Abingdon, UK). Mouse insulin was analyzed using an Ultra Sensitive Mouse Insulin ELISA Kit (Crystal Chem, IL, USA). Human WISP1 levels in sera from mice injected with Ad-WISP1 and in archived human plasma samples were determined using a Human WISP1/CCN4 PicoKine^TM ELISA Kit (Boster Biological Technology, California, USA). Specific antibodies for this ELISA were produced using T23-N367 of human WISP1 as immunogen (full-length WISP1 is 367 aminoacids). A total of 11 samples originated from clinically healthy children, aged 2–5 years, and were obtained from the Biobank of the Hospital Infantil Sant Joan de Deu. A total of 14 samples originated from clinically healthy adult men, aged 28–45 years, and were obtained from the Hospital Clinic-IDIBAPS Biobank. Informed consent was obtained for all donors. All assays were performed following the manufacturer´s instructions.