A Unisense oxygen microsensor (OX-MR) was inserted into a modified MicroRespiration chamber with an injection port (Figure 1). Cells were grown in their standard conditions and detached from the flask or petri-dish by trypsin or a scraper. Normally, 100,000-200,000 cells were used for the experiment (oxygen consumption could be measured even on 10,000 cells). To measure oxygen consumption of the cancer cells, medium containing 137 mM NaCl, 0.7 mM NaH2PO4, 5 mM KCl, 25 mM Tris-HCl pH 7.4 and 25 mg/ml ampicillin was used. The cells were permeabilized by the addition of 0.03 mg/ ml digitonin for 2 minutes and washed with the respiration medium before the measurements. Slow stirring inside the chamber was maintained and the temperature was kept at 37 °C during the measurements. When the oxygen
consumption of the cells stabilized the experiment was started.
To stimulate the pathway composed of Respiratory Complexes I+III+IV, 5 mM pyruvate and 2.5 mM malate were added, while 20 mM succinate was employed to evaluate the respiration through the Complexes II, III and IV pathways. To observe the respiration in coupled condition, 0.1 mM ADP was added after the respiratory substrates.
Conversely, to evaluate the uncoupled oxygen consumption, 5 µM nigericin plus 10 µM valinomycin were added to the cells before the addition of substrate. As respiration inhibitors, 10-50 µM rotenone to inhibit Complex I and 50-100 µM antimycin A to inhibit Complex III were employed. When the respiration was evaluated in the coupled condition, also 10-50 µM oligomycin could be used as an inhibitor of oxygen consumption. No more than 0.05 ml of the substrates were injected into the chamber.