Mitochondrial ATP Synthesis Assays

ATP synthesis rate in isolated mitochondria was measured ex-vivo in freshly isolated mitochondria by luciferin-luciferase luminescence assay in a microplate luminometer (Synergy 2 SL, BioTek, Winooski, VT, USA) as previously described [26 (link),28 (link)]. Several combinations of respiratory substrates were contemporarily tested using the following final reaction concentrations (mmol/L): 0.25 pyruvate, 0.0125 palmitoyl-L-carnitine, 2.5 α-ketoglutarate, 0.25 malate (PPKM); 0.025 palmitoyl-L-carnitine, 0.5 malate (PCM); 20 succinate, 0.1 rotenone (SR); 10 glutamate, 5 malate (GM). Measurements obtained were normalized by sample citrate synthase activity, measured spectrophotometrically as referenced [28 (link)].

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Gortan Cappellari G., Aleksova A., Dal Ferro M., Cannatà A., Semolic A., Zanetti M., Springer J., Anker S.D., Giacca M., Sinagra G, & Barazzoni R. (2020). Preserved Skeletal Muscle Mitochondrial Function, Redox State, Inflammation and Mass in Obese Mice with Chronic Heart Failure. Nutrients, 12(11), 3393.

Publication 2020

Synergy 2 SL

2 ketoglutarate Carnitine Citrate synthase Glutamate Luciferase Luciferin Luminescence assay Malate Mitochondria Pyruvate Respiratory Rotenone Succinate Synthesis

Corresponding Organization :

Other organizations : University of Trieste, Azienda Sanitaria Universitaria Integrata di Trieste, International Centre for Genetic Engineering and Biotechnology, Charité - Universitätsmedizin Berlin, Berlin-Brandenburger Centrum für Regenerative Therapien, King's College London

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Variable analysis

independent variables

Respiratory substrates
Final reaction concentrations (mmol/L): 0.25 pyruvate, 0.0125 palmitoyl-L-carnitine, 2.5 α-ketoglutarate, 0.25 malate (PPKM); 0.025 palmitoyl-L-carnitine, 0.5 malate (PCM); 20 succinate, 0.1 rotenone (SR); 10 glutamate, 5 malate (GM)

dependent variables

ATP synthesis rate in isolated mitochondria

control variables

Freshly isolated mitochondria
Sample citrate synthase activity, measured spectrophotometrically

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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