Triethanolamine hcl

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.

Cells were cultured under normoxia and hypoxia for 24 h and 5 days, and supernatant media were collected. 10 µl of the supernatant medium was added into 96-well plates in each well, and 90 μl of reagent mix (80% TRAM buffer (108 mM Triethanolamine HCl (T9534; Sigma, Burlington, MA, USA), 10.7 mM EDTA-Na2 (E4884; Merck, Burlington, MA, USA), 42 mM MgCl2 (M8266; Merck) in ddH20, pH 7.5.), 20% color reagent (1.63 mM PMS (P9625; Merck), 3.95 mM INT (I8377; Merck), 35% ethanol, and 2% Triton X-100 (T8787; Sigma-Aldrich)), 3.3 mM β-NAD (N7004; Merck) and 0.33 μl/ml LDH (L2500; Merck)) was added. Plates were incubated for 7 min at RT avoiding light. Optical density was measured in a plate reader at 490 nm and lactate was quantified against a lactate standard curve (#71718; Fluka Chemika, Buchs, Switzerland) using the abc-formula (ax² + bx + c = 0).