Cyclic gmp eia kit

MPEC lysates were extracted in the presence of 3-isobutyl-1-methylxanthine (IBMX; 1 mmol/L) by addition of 0.1 mol/L HCl. cGMP concentration was assessed following acetylation using Cyclic GMP EIA Kit (Cayman Chemicals) according to the manufacturer's instructions. Resulting cGMP concentrations were normalized to protein content per sample.

Quantitative assays for cGMP were performed using a commercial enzyme immunoassay kit (Cayman Chemical Cyclic GMP EIA kit, Ann Arbor, MI, USA). For penile cGMP content, frozen penile tissue was homogenized in 5% trichloroacetic acid and centrifuged. TCA was extracted from the supernatant with three washes of water-saturated ether. cGMP was expressed as pmol/mg tissue.

Murine blood was drawn from the retro-orbital plexus of 12- to 20-week-old mice under anesthesia without reawakening into 7xACD buffer (in mM: 85 sodium citrate, 72.9 citric acid, 110 d-glucose). To obtain platelet-rich plasma, blood was centrifuged at 250×g for 2 min at room temperature. Then, the supernatant was centrifuged at 2000×g for 2 min. Pelleted cells were washed in platelet wash buffer (in mM: 4.3 K₂HPO₄, 4.3 Na₂HPO₄, 24.3 NaH₂PO_4, 113 NaCl, 5.5 D-glucose, pH 6.5) supplemented with 0.1% BSA and resuspended at a density of 6.4 × 10⁸ mL⁻¹ in platelet Tyrode buffer supplemented with 0.1% BSA. Tubes containing suspensions of washed platelets in Tyrode buffer (5 × 10⁷ platelets per sample) were placed at room temperature in racks and incubated with 50 nM DEA/NO for 15 s without vortexing (static condition) or on a vortex shaker (Barnstead Thermolyne Maxi Mix II Vortex Shaker, set to full speed). Control platelets were allowed to rest without DEA/NO and vortex treatment. Then, ice-cold ethanol was added to a final concentration of 66% ethanol. Tubes were put on ice for 15 min and then centrifuged at 18,000×g for 10 min at 4 °C. Supernatants were dried at 90 °C and the cGMP content was determined using an enzyme immunoassay kit (Cyclic GMP EIA Kit, Cayman Chemical) according to the manufacturer’s protocol.

Mice were dissected immediately or 10 min after intraperitoneally (IP) injection of 60 mg kg⁻¹ DETA-NO. Harvested organs were first powdered at −70 °C (Retsch MM301) and subsequently homogenized in 1 ml ice-cold 100% ethanol. Extracts were centrifuged at 14,000g for 10 min at 37 °C. The supernatant was transferred and the pellet was washed once with 0.5 ml ice-cold 100% ethanol and centrifuged at 14,000g for 10 min at 37 °C. The total supernatant was dried under vacuum at 30 °C. The pellet was redissolved in protein buffer (20 mmol l⁻¹ HEPES, 350 mmol l⁻¹ NaCl, 0.5 mmol l⁻¹ EDTA, 20% glycerol, 0.5% Triton X-100, EDTA-free protease inhibitor mix), centrifuged for 10 min at 14,000g at 37 °C and protein concentration of the supernatant was measured using a BCA Protein Assay Kit (Pierce). cGMP pellets were dissolved in EIA-buffer and cGMP concentrations were measured using the acetylation-protocol, of the Cyclic GMP EIA Kit (Cayman Chemical). cGMP concentration is expressed as picomole cGMP per milligram of protein.

The role of AERV on cyclic guanosine monophosphate (cGMP) levels was evaluated according to Estancial et al. [26 (link)]. Aortic rings from 2K1C rats (2–3 mm; n = 5) were removed and placed in an organ bath with Krebs–Henseleit solution (composition in mm: 4.7 KCl, 117 NaCl, 1.2 MgSO₄, 2.5 CaCl₂, 1.2 KH₂PO₄, 25 NaHCO₃, and 11 glucose) at 37 °C with 95% O₂ and 5% CO₂. Aortic rings were incubated with SNP (10 μm), or AERV at the concentrations of 0.001, 0.003, and 0.01 mg/mL for 15 min in the presence or absence of ODQ (100 μm, 30 min). From tissues samples the supernatants were collected after their removal and homogenization. The intracellular cGMP levels were determined as described by the manufacturer (Cayman Chemical Cyclic GMP EIA kit, Ann Arbor, MI, USA). All experiments were performed in triplicate.