An Improved Assay for Measuring Low Levels of Nitric Oxide in Cultured Pulmonary Myofibroblasts

Abstract

Quantification of nitric oxide (NO) from cultured cells is a valuable tool for studying cell signaling. Detection of NO in biological fluids can be difficult however, due to its transient half-life and low physiological concentrations. In this study, we have refined an existing amperometric method to determine relative levels of accumulated nitrogen oxides (NO X ) in cell culture and have used this method to reproducibly quantify NO from cultured pulmonary myofibroblasts. Basal levels of NO produced by pulmonary myofibroblasts ranged from 0.6 nM to 20 nM and varied due to the growth conditions of the cells, i.e. higher NO concentrations were observed in differentiated cells. The constitutive eNOS isoform is primarily responsible for the observed NO accumulation in these cells since transcript levels of eNOS are 10-fold higher than the inducible iNOS form while nNOS was undetectable. Treatment of myofibroblasts with the inhibitors L-NNA and L-NAME resulted in a concentration dependent decrease in measured NOx. Overall, the improved assay presented here should be applicable to measuring NOX levels from many different cell types and under a wide variety of conditions.

Investigation of Some Medicinal Plants Inhibitory Effect on NO Production in Oxidative Stressed PC12 Cells

“…Cytotoxic effect was not detected in the unstressed cells either in the present or in the previous study on this extract, however, it lowered the cell viability during the stress condition. 28 Consequently, C. commutatus extract might be effective in other biological studies related to the suppression of NO production. According to our results, extract of S. multicaulis inhibited NO production best at higher doses, and protected cells at lower doses.…”

“…Experiments were performed in serum-free DMEM to avoid rapid H2O2 degradation by antioxidants and interference with Griess assay by FBS. 27,28…”

Study on constituents of Scutellaria nepetifolia as a potent source of phytochemicals with NO inhibitory effect

“…The dilution of the stock solutions and hydrogen peroxide (H2O2) to gain interested concentrations were carried out with DMEM prior to use. All of the experiments involving exposure to H2O2 and measurement of nitric oxide (NO) were performed in serum-free DMEM to avoid rapid H2O2 degradation by antioxidants and interference with Griess assay by FBS (Clément et al 1998;Sharma et al 2014).…”

Nitric oxide detection methods in vitro and in vivo

“…NO selective electrode allowed robust and reproducible detection of subtle changes in NO concentration produced by cultured pulmonary myofibroblasts of rats under wide variety of conditions. 88 Recently, amperometric NO release was viewed in real time at the stimulated acupoints in rats inserted with an acupuncture microsensor needle made up of gold film and doped with iron-porphyrin functionalized graphene composite. 89 Further, the authors claimed the application of the acupuncture needle to detect other vital signaling molecules ( Figure 11 ).…”