Mitochondrial Respiration Complex Assay

Cells were rinsed twice in ice-cold PBS, lysed with 0.5 mL buffer A (50 mmol/L Tris, 100 mmol/L KCl, 5 mmol/L MgCl₂, 1.8 mmol/L ATP, 1 mmol/L EDTA, pH 7.2) containing the protease inhibitor cocktail III [100 mmol/L AEBSF, 80 mmol/L aprotinin, 5 mmol/L bestatin, 1.5 mmol/L E-64, 2 mmol/L leupeptin and 1 mmol/L pepstatin (MerckMillipore, Milan, Italy) 1 mmol/L PMSF, 250 mmol/L NaF]. Samples were centrifuged at 650× g for 3 min at 4 °C, supernatants were transferred into a new tube series and centrifuged at 13,000× g for 5 min at 4 °C. The supernatants were discarded, the pellets containing mitochondria, after a washing step with 0.5 mL buffer A, were re-suspended in 0.25 mL buffer B (250 mmol/L sucrose, 15 μmol/L K₂HPO₄, 2 mmol/L MgCl₂, 0.5 mmol/L EDTA, 5% w/v BSA). 50 μL were sonicated and used for protein quantification. The activity of mitochondria respiration complexes was evaluated according to [18 (link)]. Results were expressed as nmol red cit c/min/mg mitochondrial proteins.

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Roux C., Mucciolo G., Kopecka J., Novelli F., Riganti C, & Cappello P. (2021). IL17A Depletion Affects the Metabolism of Macrophages Treated with Gemcitabine. Antioxidants, 10(3), 422.

Publication 2021

bestatin leupeptin pepstatin

Aebsf Aprotinin Bestatin Buffer Cells Cold Edta K2hpo4 Leupeptin Mgcl2 Mitochondria Mitochondrial proteins Pellets Pepstatin Protease iii Protein Respiration Sucrose Tris

Corresponding Organization : University of Turin

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Activity of mitochondria respiration complexes

control variables

Cells were rinsed twice in ice-cold PBS
Cells were lysed with 0.5 mL buffer A (50 mmol/L Tris, 100 mmol/L KCl, 5 mmol/L MgCl2, 1.8 mmol/L ATP, 1 mmol/L EDTA, pH 7.2) containing the protease inhibitor cocktail III
Samples were centrifuged at 650× g for 3 min at 4 °C
Supernatants were centrifuged at 13,000× g for 5 min at 4 °C
Pellets containing mitochondria were washed with 0.5 mL buffer A and re-suspended in 0.25 mL buffer B (250 mmol/L sucrose, 15 μmol/L K2HPO4, 2 mmol/L MgCl2, 0.5 mmol/L EDTA, 5% w/v BSA)

controls

None explicitly mentioned

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