2.4. Determination of Mitochondrial ROS Production Using Redox Sensitive GFP

To measure the production of ROS in hypoxia, we used the ratiometric redox-sensitive biosensor RoGFP (redox sensitive GFP) targeted to either the mitochondrial matrix or the inter membrane space [54]. The advantage of using this probe is that the oxidized and reduced state of the protein is measured at two excitation wavelengths, 405 and 495 nm, and emission at 510 nm independently of the expression levels. The ratio of fluorescent 405/495 is directly proportional to the levels of ROS [54]. Plasmids (1 μg) expressing the targeted RoGFP to either the mitochondria matrix (matrix-RoGFP, Addgene plasmid #49437, Watertown, MA, USA) or the intermembrane space (GPD-RoGFP, Addgene plasmid #49436, Watertown, MA, USA) [54] were used to transfect controls, RISP and COX10 KO fibroblasts using the GenJET transfection reagent (SignaGen Laboratories, Frederick, MD, USA) as per manufacturer instructions. After 24 h of transfection, cells were exposed to either normoxia or 1% oxygen for 24 h. After hypoxia incubation, cells were fixed inside the hypoxic chamber with 4% PFA for 15 min and washed with PBS 3 times as previously described [55]. Fixing cells was performed to avoid changes in fluorescence due to re-oxygenation after hypoxic treatment when processing cells for microscopy. Fluorescence 405/495 ratio was the same in cells fixed or not fixed under normoxic conditions, and fluorescence did not change in fixed cells after the addition of 10 mM DTT (Sigma D9779, St Louis, MO, USA) or 10 mM tBOH (Sigma B2633, St Louis, MO, USA). Fluorescence was recorded using a Leica SP5 multiphoton upright confocal microscope at two excitation wavelengths of 405 and 495 nm and emission wavelength of 510 nm. Fluorescent intensities at the two excitation wavelengths for each cell were calculated using ImageJ software and the ratio 405/495 was calculated as a measurement of redox state of the cells. Fluorescence ratios for at least 20–30 cells per experiment were imaged and independent experiments were done at least 3 times. Data from all independent experiments were combined.