Foxy probe

Cells were subjected to ischemia reperfusion by exposing them to oxygen-glucose deprivation/reoxygenation (OGDR). Briefly, DMEM (glucose and serum free) that had been effervesced with 5% CO₂ and 95% N₂ for 1 h was applied to the cells. Cells were then placed inside a sealed chamber with an atmosphere of 5% CO₂ and 95% N₂ for 1.5 h, followed by re-oxygenation in fresh OptiMEM+ at 37°C in 5% CO₂ and 95% air for 0, 1.5, 4 or 24 h. Oxygen levels in the media were measured using a fibre optic oxygen (FOXY) probe (Ocean Optics).

The oxygen content was measured through a fibre optic oxygen sensor probe, (FOXY probe, ocean optics, USA), connected to a MultiFrequency Phase Fluorometer (Ocean Optics, Florida, USA). The oxygen sensor was pre-calibrated by curves generated from standards concentration values of O ₂ dissolved in anoxic and normoxic solutions. The second order polynomial algorithm was used for better curve fitting and for accurate oxygen measurements in a broad oxygen concentration range. The continuous visualization of the O ₂-concentration values were achieved by a software program (OOISensors Oxygen Measurement Software, Ocean Optics, USA) installed on a personal computer (PC) connected to the oxygen sensor system, see Figure 1 above.

Oxygen evolution experiments were performed in a 6.7 mL headspace Schlenk tube sealed with a rubber septum (PFTE). The Schlenk tube was covered with aluminum foil, in order to avoid an early light-induced reaction of the system, and filled with 1 mM (9.4 mg) [Ru(bpy)₃]Cl₂, 5 mM (14.9 mg) Na₂S₂O₈, the desired amount of catalyst, and 12.5 mL of 40 mM KP_i buffer solution at pH 7.0. Experiments employing the RuCo₉ salt as catalyst were performed with and without addition of [Ru(bpy)₃]Cl₂, the former for comparison in the same conditions required for Co₃O₄ experiments. Suspensions were completely deaerated by purging with nitrogen. A baseline of 20 min was recorded to ensure that no oxygen leakage or side reactions took place. Next, the system was exposed to the light of a blue LED lamp (wavelength at peak emission = 465 nm; OSRAM Opto Semiconductors) working at 0.20 A and 11.4 V. The concentration of oxygen in the headspace was measured by employing a O₂-sensor probe (Ocean Optics NeoFOX oxygen-sensing system equipped with a FOXY probe). Turnover number (TON) and turnover frequency (TOF) were estimated per Co₉ content as obtained from chemical analyses on fresh compounds (see SI).