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Inhibitor concentration is too low or too high. Very low doses of inhibitor may not affect ROS production by inducer. Alternatively, very high doses of the inhibitors may cause oxidative stress itself and generate fluorescent signal. Optimize the concentration of the inhibitor and pretreatment time for each particular cell line.
Inappropriate time point for detection. Inhibitors may be loaded into cells for too long a time or, when inducer concentrations are too high, the inhibitor becomes insufficient. Optimize detection times and inducer concentrations.
Inappropriate filter set on the microscopy. Use correct filter for each fluorophore.
- Oxidative stress detection requires a filter set compatible with Fluorescein (Ex/Em: 490/525nm).
- Superoxide detection requires a filter set compatible with Rhodamine (Ex/Em: 550/620nm).
- NO detection requires a filter set compatible with Cyanine 5 (Ex/Em: 650/670nm).