In-vitro generation of oxidative, nitrosative and peroxynitrile stress

For the in-vitro generation of oxidative stress (ROS), nitrosative stress (RNS) and peroxynitrile stress, menadione, SNAP (S-nitroso-N-acetylpenicillamine) or mixture of both reagents were used, respectively. The sub-lethal dose of all the stress inducing reagents were previously determined [23] and doses of 5 μM menadione for oxidative stress, 300 μM SNAP for nitrosative stress and mixture of 2.5 μM menadione and 250 μM SNAP for peroxynitrile stress were used in this study. The sub-lethal doses of different stress inducing agents were determined by studying the changes in percent cell viability using MTT assay according to the manufacturer’s protocol (In-Vitro Toxicology Assay kit, MTT Based, Sigma, USA).