Methodology for the analysis of BMP9 content inserum. C2C12BRE cells were treated as indicated in Figure 1 and were stimulated with 10% FCS in the absence or presence of 0.1 ng/ml BMP9 antibody. Luciferase activities were normalised to protein content and relative luciferase units (RLU) were calculated following subtraction of background values obtained from unstimulated cells. The mean decrease of RLU in the presence of BMP9 antibody was calculated (Δ luciferase units). In the same experiment C2C12BRE cells were treated with increasing concentrations of BMP9 within the linear range in order to calculate a standard curve for BMP9 activity as described in Figure 2. The equation of the line was solved and the relative amount of BMP9 calculated using the Δ luciferase value. To obtain total BMP9 concentration per ml of serum this value is multiplied by the dilution factor of 20. Data shown is a representative experiment performed in quadruplicate ± SD.
Herrera and Inman BMC Cell Biology 2009 10:20 doi:10.1186/1471-2121-10-20