Measuring Mitochondrial Stress in Serum

Levels of MIS in serum samples were evaluated by measuring intracellular ATP content (MIS-ATP) and ROS generation (MIS-ROS) as described^{12 (link),13 (link)}. pRL-mTK-transfected mouse Hepa1c1c7 cells (5 × 10⁴/well) in a 96-well plate were treated with 10 μL heat-inactivated-serum samples for 48 h. The ATP content was determined using the CellTiter-Glo luciferase kit (Promega, Madison, WI, USA), with the output being normalized to Renilla luciferase activity. ROS level was determined using 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate and acetyl ester (CM-H₂DCFDA; Molecular Probes, Eugene, OR, USA). Both MIS-ATP and MIS-ROS were expressed as % of charcoal stripped serum (CSS)-treated control. The intra- and inter-assay coefficients of variation for these methods were less than 6.0%.