Dea no

The determination of sGC activity was performed in homogenates of mouse stroked ipsilateral cortex and basal ganglia vs. non-stroke cortex and basal ganglia, as previously described.^{23 (link)} Briefly, crude brain homogenates of stroked and non-stroked C57Bl/6 mice were measured as the formation of cGMP at 37 C during 10 min in a total incubation volume of 100 μl containing 50 mM triethanolamine–HCl (pH 7.4, Sigma), 3 mM MgCl, 3 mM glutathione (Carl Roth), 1 mM 3-isobutyl-1-methylxanthine (IBMX, Enzo LifeSciences), 100 mM zaprinast (Enzo LifeSciences), 5 mM creatine phosphate (CalBiochem), 0.25 mg/ml creatine kinase (CalBiochem), and 500 mM GTP. The reaction was started by simultaneous addition of the crude brain homogenates and either DEA/NO (Enzo LifeSciences) or BAY58-2667 (Adipogene), respectively. After incubation of each sample (n = 3 each per group) for 10 min the reaction was stopped by boiling for 10 min at 95 °C. Thereafter, the amount of cGMP was subsequently determined by an enzyme immunoassay (ENZO cGMP EIA kit) using different sample dilutions in the linear range.

Carboxyfluorescein succinimidyl ester for T-cell division analysis and 1-MT, an IDO inhibitor, were purchased from Sigma-Aldrich (St. Louis, MO, USA). DEANO, a NO donor, was purchased from Enzo Life Science (East Farmingdale, NY, USA). L-NMMA, a NO inhibitor, was purchased from EMD Millipore (Billerica, ME, USA). 4EGI-1 was purchased from Merck Millipore (Billerica, ME, USA). Lymphocytes were cultured with 1 mM L-NMMA, 1 nM 1-MT, 1–2 mM DEANO, or 20 μM 4EGI-1. CD4-FITC (RM-4-5), CD8-PE (53-6.7), CD25-APC (3C7), CD122-PE (TM-1), CD132-PE (4G3), Annexin-V-FITC, and propidium iodide were purchased from BD Biosciences (San Jose, CA, USA). RNasin was purchased from Promega (Madison, WI, USA).