Nitrate nitrite fluorometric assay

Mice were fasted overnight and retro-orbital blood was drawn beginning at 11:00 a.m. the following morning to assess steady-state levels of plasma insulin (Mouse Ultrasensitive ELISA kit, Alpco, Salem, NH), C-peptide (ELISA, Alpco), non-esterified fatty acids (NEFA C kit, Wako Diagnostics, Richmond, VA), triacylglycerol (Pointe Scientific Triglyceride), endothelin-1 (ELISA kit, ab133030, Abcam, Cambridge, MA), TNFα (SimpleStep ELISA kit, ab208348, Abcam) and IL-6 (ELISA Kit, ab222503, Abcam). Hepatic triacylglycerol was measured as previously described [26 (link)], hepatic nitric oxide (NO) levels using Nitrate/Nitrite Fluorometric Assay (780051, Cayman Chemical, Ann Arbor, MI), and hepatic levels of reduced glutathione (GSH) were measured using the Bioxytech GSH-400 kit (OXISResearch, Portland, OR). Plasma Alanine Transaminase (ALT) (ab105134, Abcam) and Aspartate Aminotransferase (AST) (ab105135) colorimetric assays kits were used to assess liver function.

The nitrate/nitrite fluorometric assay (780051) from Cayman (Ann Arbor, MI, USA) was used as indirect measure for NO production. Therefore, approximately one third of a RF muscle per mouse was homogenized in 5 ml PBS, pH 7.4 at 4°C. After quantification of the protein concentration in aliquots, NOS activity was initiated by addition of the cofactor mixture to 1 mg of homogenate protein at RT and stopped after 15 min by centrifugation through ultrafiltration tubes (UFC703008, Centricon Plus-70, Merck, Darmstadt, Germany). Aliquots of the supernatants collected at initiation and termination of the reaction as well as nitrate standards were subjected in duplicates to the nitrate/nitrite fluorometric assay according to the instructions of the manufacturer. The extinction of the samples was recorded at an excitation wavelength of 360-365 nm and an emission wavelength of 430 nm in a SpectraMax 190 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) and used to calculate the rates of nitrate/nitrite production per time unit and mg of homogenate protein.

Active caspase-1 was detected in living ECs with use of the Fluorescent Labeled Inhibitor of Caspases (FLICA) caspase-1 assay kit (ImmunoChemistry Technologies). FLICA-FAM-YVAD-FMK is a cell-permeable, non-toxic fluorochrome inhibitor of caspase-1 that interacts with the enzymatic-reactive center of activated caspase-1 via the YVAD recognition sequence, thus forming a covalent thioether adduct with the enzyme through the FMK moiety. The resulting green fluorescence is a direct measure of caspase-1 activity, which was analyzed by FACS with 488-nm excitation and 530-nm emission. NO was detected as the accumulated nitrite/nitrate, the stable breakdown product of NO, in cell culture media by use of a nitrate/nitrite fluorometric assay (Cayman Chemicals).