Basal and stimulated intracellular cGMP concentrations were evaluated in primary TG cultures after 24 h in vitro. A commercial enzyme-linked immunosorbent assay (ELISA) kit for cGMP (MBL International Corporation, Woburn, MA, USA) was used, following the manufacturer’s instructions as previously described.23 (link) Protein concentrations in cell lysates were determined by the bicinchoninic acid method (Sigma). The cGMP concentrations in pmol/µg of protein were extrapolated from a best-fit line calculated from serial dilutions of a cGMP sample standard.
To determine BNP concentration in medium samples, a commercial ELISA kit for mouse BNP was used (Abnova, Hidelberg, Germany) following the instructions of the manufacturer, as previously reported.23 (link) After 24 h from plating cells, culture medium was collected. All samples were centrifuged at 1200 r/min for 5 min, and the supernatants immediately processed for BNP measurement. Protein concentrations in cell lysates were determined by the bicinchoninic acid method (Sigma). All samples were run in triplicate and values averaged.
To determine BNP concentration in medium samples, a commercial ELISA kit for mouse BNP was used (Abnova, Hidelberg, Germany) following the instructions of the manufacturer, as previously reported.23 (link) After 24 h from plating cells, culture medium was collected. All samples were centrifuged at 1200 r/min for 5 min, and the supernatants immediately processed for BNP measurement. Protein concentrations in cell lysates were determined by the bicinchoninic acid method (Sigma). All samples were run in triplicate and values averaged.
