Adi 900 014

CSF levels of cAMP and cGMP were determined by ELISA. Both cAMP and cGMP ELISAs were obtained from Enzo Lifesciences (cAMP: #ADI-900-066; cGMP: #ADI-900-014) and used according to the manufacturer’s protocol. Briefly, for cAMP, 100 μl standard or CSF and 100 μl 0.1 M HCl were added into the appropriate wells. For cGMP, 200 μl CSF and 200 μl 0.1 M HCl were used. After adding 50 μl of the conjugate and 50 μl of the antibody to the appropriate wells, the plates were incubated at room temperature for 2 h with shaking. After washing steps, pNpp substrate and stop solution were added and the optical density was measured at 405 nm. The assays were blind for patient identification and disease status.

To measure the pulmonary release of cGMP, pulmonary arterial blood was collected in sample tubes coated with traces of heparin. The mixture was centrifuged at 3,000 rpm for 20 min, at 4 °C to obtain the plasma. The level of cGMP in pulmonary arterial plasma was determined by radioimmunoassay using a direct cGMP EIA kit according to the manufacturer’s instructions (ADI-900-014, Enzo Life Sciences, USA).

The cGMP levels in the glioblastoma cell lines were measured using an ELISA kit according to the manufacturer's instructions (ADI-900-014, Enzo Life Sciences, Italy). cGMP concentration was normalized per mg of total proteins.