The following recommendations can help to obtain better measurements:

1. The use of highly purified ion-free water is recommended for preparation of all solutions.

2. The pH of the incubation buffer (7.4) should be carefully checked, and it should be readjusted if needed, since ROS production can be affected by this parameter.

3. Excessive mitochondrial concentration during the incubation at 37°C can cause decreases in fluorescence and should be avoided.

4. A chemical (in the absence of mitochondria) H2O2 pulse experiment can be easily performed each day before starting the measurements with mitochondria, or even before sacrificing the animal to isolate them. This reaction can be very useful to check the quality of the horseradish peroxidase and homo-vanillic acid reactants, in order to avoid erroneously attributing an absence or very low rate of ROS production to the mitochondrial preparations if a recently received or stored reactant is not in good condition or has lost enzymatic activity. For this purpose H2O2 is added from a standard H2O2 solution to the homovanillic-HRP detection system, and the occurrence of the corresponding increase in fluorescence is checked.

5. The use of soap to clean the materials (glassware, etc.) that will be in direct contact with the mitochondria should be avoided.

6. Tubes with isolated mitochondria must be kept on ice and at high mitochondrial concentrations. The mitochondria must be used between 2 and 3 hours after isolation. If longer time periods are needed, it is necessary to check again the functionality of the mitochondria (RCI) after the assays.

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