Igf 1

Cells were incubated with I^125-labeled insulin or IGF-1 (PerkinElmer, Waltham, MA, USA) with indicated concentration of unlabeled insulin or IGF-1 on ice for 5 h. After washing with cold PBS for three times, the cells were lysed with 4% NaOH for 1 h at room temperature. Then the cell lysis was transferred to the scintillation bottle, and the radioactivity was measured by a scintillation counter.

Serum samples were analyzed in duplicate for free/bioactive IGF-1 (Diagnostic Systems Laboratories, Webster, TX), and free testosterone (Alpha Diagnostic International, San Antonio, TX) using an ELISA (enzyme linked immunoabsorbent assay). For IGF-1, assay sensitivity is 0.06 ng/ml, and does not cross-react with albumins or GH (growth hormone) binding proteins. For testosterone, sensitivity is 0.17 pg/ml. For IGF-1 and testosterone the subsequent absorbances, which were directly proportional to the concentration of analyte in the sample, were measured at a wavelength of 450 nm using a microplate reader (Wallac Victor 1420, Perkin Elmer, Boston MA). A set of standards of known concentrations for IGF-1 and testosterone were utilized to construct standard curves by plotting the net absorbance values of the standards against their respective protein concentrations. By applying a four-part parameter curve using MikroWin microplate data reduction software (Microtek Lab Systems, Germany), the free IGF-1 and free testosterone concentrations in the serum samples were calculated. The overall intra-assay percent coefficient of variation was 4.9 and 3.7 % for IGF-1 and testosterone, respectively.