Nitric oxide analyzer

Primary renal tubular epithelial cells (RTEC) from C57BL/6J mice (Cell Biologics, Chicago, IL) were cultured as per manufacturer’s instructions. RTEC (1x10⁶ cells/well) were treated with IL-17A (50 or 200 ng/ml) or TNFα (5 ng/ml) or IL-17C (50 or 200 ng/ml) or IL-17F (50 or 200 ng/ml) and IL-17 and TNFα in combination for 24 h. Recombinant murine IL-17A, IL-17C, IL-17F and TNFα were purchased from Peprotech (Rocky Hill, NJ).
Bone marrow derived macrophages (BMDM) from C57BL/6J mice were cultured for 7 days in the presence of L929 supernatants. BMDM and RTEC (1x10⁶ cells/well) were treated with bradykinin (R&D Biosystems, Minneapolis MN) or left untreated for 24 h. LPS (Sigma Aldrich, St Louis, MO) was used as positive control. Supernatants were subjected to analyses using commercially available IL-6, IL-1β and TNFα ELISA kits (Ebiosciences, Dallas TX. Nitrite concentrations were measured by tri-iodide based reductive chemiluminescence as previously described [57 (link)]. Briefly, samples were injected into tri-iodine to reduce nitrite to NO gas that was detected by a Nitric Oxide Analyzer (Sievers, GE).

Nitrite and nitrate were detected with Nitric Oxide Analyzer (NOA 280i, GE, USA) with nitrogen as the carrier gas. Reagent of 0.011 g/mL iodine ion was applied for nitrite detection and 0.008 g/mL acidic vanadium trichloride solution was used for nitrate detection. The standard curve was generated via measurement of duplicate injections of each standard solution with R² > 0.999. Each urine specimen was injected three times with the volume of 100 μL (25 (link), 26 (link)).