3 isobutyl 1 methyl xanthine (ibmx)

Cholera toxin, the JAK2 inhibitor (Tyrphostin AG490 (AG490), the PI3K inhibitor (LY-294002), ACTH, and the PDE3B inhibitor (IBMX), were purchased from Sigma Aldrich (St-Louis, MO). The inhibitors, AG490, LY-294002, and IBMX, were respectively diluted in DMSO for stock solutions of 1mg/ml, 5mg/ml, and 0.5 M. A G-CSF receptor chimera (Fc Chimera Active) was purchased from Abcam (Cambridge, MA), and G-CSF was obtained from Loma Linda University Pharmacy; both were diluted in phosphate buffered saline. The following concentrations were used: cholera toxin (50 ng/ml) (Hsu et al, 2006 (link)); AG490 (50 μM) (Chen et al, 2005 (link)); LY-294002 (20 μM) (Williams et al, 2010 (link)); and IBMX (10 μM) (Montero-Hadjadje et al, 2006 (link)). A dose-response study of G-CSF (using 30, 100, and 300 ng/ml) (Hsu et al, 2006 (link)), and the G-CSF receptor chimera (at 10, 30, and 100 ng/ml) after cholera toxin treatment was conducted. At 24 hours, growth media was collected from each well for subsequent assays and analysis. G-CSF-treated cells were collected at the following time points after treatment initiation: 0, 5, 15, 30, 60, and 120 minutes, to determine the activity of the JAK/PI3K/Akt/PDE3B pathway.