Effect of incubation time on BMP assay sensitivity. C2C12BRA cells were plated at 4 × 103 cells/well. Recombinant BMP-4 at the indicated concentrations was incubated with the cells for 5, 10, 15 or 24 h prior to assaying for luciferase activity. Background luciferase level in the control sample (no BMP) was subtracted from each of the experimental values. The results are expressed as relative luciferase units (RLU) (the activity with 14 pM of BMP-4 after 10 h incubation time equals one). Each point represents the mean ± SEM of triplicate wells from one representative experiment. Similar results were obtained in two separate experiments.