Figure 5

LPS induced NO resistance in differentiated RAW 264.7 macrophages is abrogated by chemical inhibitors affecting the intra-cellular redox protection/detoxification systems. RAW 264.7 cells were first treated in vitro, as previously, culturing them in medium (undifferentiated cells), or medium supplemented with 5 μg/ml LPS. After for 24 hours, surviving-LPS-differentiated RAW 264.7 macrophages, and control undifferentiated RAW 264.7 cells, were washed and incubated for an additional 2 hours period in the presence of the redox inhibitors BSO (an inhibitor of γ-glutamyl cysteine synthase), or ATZ (a catalase inhibitor), or DETC (an inhibitor of both Mn SOD and Cu/Zn SOD). Subsequently, the resistance of the treated cells to exogenous NO was evaluated by culturing them (or not) in the presence of 1 mM DETA-NO (a dose previously established to be toxic for undifferentiated RAW 264.7 cells, Figure 2A). The % viability of cell exposed to exogenous NO was assessed after 48 hours as the percent of metabolically active cells reducing formazan (MTT test) compared to control (number of RAW 264.7 cells undifferentiated, not exposed to exogeneous NO, surviving at the end of the culture period = 100% cell viability).