Quantitation of Nitric Oxide in Cell Culture by Luciferin-Luciferase Chemiluminescence

Abstract

The chemiluminescent method is proposed for quantitation of NO generation in cell cultures grown on standard 96-well plates. The method is based on activation of soluble guanylyl cyclase by NO; the product of guanylyl cyclase reaction, pyrophosphate, after conversion to ATP by ATP sulfurylase, is detected in luciferin-luciferase system. The method has been applied to the measurement of NO generation by activated RAW 264.7 cells, primary bone-marrow murine macrophages and aortic endothelial cells. For macrophages, activated by lipopolysaccharide and γ−interferon, the rate of NO production is about 100 amol/(cell·min); the same rate was found from the measurements of nitrite, the final product of NO oxidation, for the same cells. For endothelial cells, the basal rate of NO generation is 5 amol/(cell·min); it approximately doubles upon activation by bradykinin, Ca2+ ionophore A23187 or mechanical stress (shaking). For both types of cells the measured NO generation is strongly affected by inhibitors of NO synthase. The sensitivity of the method is about 50 pM/min, allowing to measure NO generated by 102 – 104 cells. The direct comparison shows the chemiluminescent method to be two orders of magnitude more sensitive than fluorescent detection using DAF-FM.