Detection of endogenous S-nitrosothiol formation in activated macrophages. J744A.1 cells were treated without (control) or with LPS plus INF-γ (LPS/INF-γ) overnight to induce iNOS expression. W1400 was added to inhibit iNOS activity as indicated. A. Cells were rinsed three times in ice cold HBSH, scraped and sonicated in 40 mM of NEM. Nitrosothiol context of the extracts was analyzed by the ozone-based chemiluminescence as described in methods. B. After activation, cells were washed, fixed, blocked with NEM, reduced by 2 mM PE SO3 and labeled with Alexa Fluor 488 C5 maleimide.