Oxygen biosensor system

To determine the extent of mitochondrial dysfunction, freshly isolated mitochondria from experimental mice were used for the measurements of mitochondrial permeability transition (MPT), ATP levels and O₂ consumption followed by previously published procedures (Moshal et al., 2008 (link); Wang et al., 2005 (link); Wilson-Fritch et al., 2004 (link)).
To detect MPT, isolated mitochondria were resuspended in a medium containing (in mmol/l): 180 KCl, 10 EDTA, and 10 HEPES, pH 7.4, with 0.5% BSA. To remove EDTA and BSA, the mitochondrial pellet was washed two times with wash buffer (in mmol/l: 180 KCl and 10 HEPES, pH 7.4). Mitochondrial swelling was determined by a decrease in light absorption at 540 nm with 250 μg of mitochondrial protein in swelling buffer containing (in mmol/l) 250 sucrose, 10 Tris-morpholinosulfonic acid, 0.05 EGTA, pH 7.4, 5 pyruvate, 5 malate, and 1 phosphate by the method described elsewhere (Rodrigues et al., 2002 (link)). The MPT was measured spectrophotometrically before and after the addition of CaCl₂ (250 μmol/l).
The levels of ATP in isolated mitochondria were measured by using the ATP Determination Kit (Invitrogen; Catalog No. A22066) according to the manufacturer’s recommendations.
Mitochondrial O₂ consumption was determined by using the BD Oxygen Biosensor system (BD Bioscience, Bedford, MA) as described earlier (Wilson-Fritch et al., 2004 (link)).

Oxygen consumption rates in cells were conducted in an Oxygen Biosensor System (BD). Oxygen consumption rates were measured over 3 hours every 15 minutes using a fluorescent plate reader.