Figure 1. Response of ACTOne PDE4 cell line & parental cell line to Rolipram and Ro20-1724. ACTOne PDE4 cells and parental cells (AG-10200-200) were plated overnight in 20 ml culture medium on a 384 well Biocoat plate. The next day, cells were dye-loaded with 20 ml/well of ACTOne membrane potential dye. After 2 hour of incubation at room temperature, baseline was recorded using a FlexStation (Molecular Devices) (F0). 10 ml of PDE inhibitors at various concentrations were added to the cell plate, and the data was recorded 30 minutes (Ft) after drug addition. Dose response curves were generated by Prism. A. Dose response curves of Rolipram and Ro20-1724 in ACTOne PDE4 cell line. EC50 = 0.28 mM for Rolipram and EC50 = 2.34 mM for Ro20-1724. B. Parental cells do not respond to Rolipram or Ro20-1724.