Mitochondrial complex activity assays
michela.deleidi, Maria Jose Perez J.
Abstract
Mitochondria complex activity assays measure the activity levels of the different complexes of the mitochondrial electron transport chain (ETC).
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Mitochondrial complex activity assays
Isolate mitochondria from HEK cells, iPSC-derived neurons, or midbrain organoids using the Qproteome Mitochondrial isolation kit (QIAGEN, Cat. No. / ID: 37612) according to manufacturer's instructions.
Measure Complex I (NADH oxidase/coenzyme Q reductase) using the MitoCheck Complex I Activity Assay kit (Cayman Chemical, cat# 700930).
Determine the rate of NADH oxidation, which is proportional to CI activity, by a decrease in absorbance at 340 nm over 0h 15m 0s in the presence of ubiquinone and potassium cyanide to inhibit complex IV and prevent oxidation of ubiquinone.
To this end, resuspend 3mg of purified fresh mitochondria in 200µL of MAS buffer (70millimolar (mM) sucrose, 220millimolar (mM) mannitol, 5millimolar (mM) KH2PO4, 5millimolar (mM) MgCl2, 1millimolar (mM) EGTA, 2millimolar (mM) HEPES 7.4) and seed in XFpSeahorse microplates.
Centrifuge the plate at 2000x g,4°C.
Measure the OCR before and after the serial addition of pyruvate + malate (5millimolar (mM) each) + ADP 3,5 mM or 1 mM Succinate + 4micromolar (µM) rotenone, 4micromolar (µM) rotenone + 8micromolar (µM) antimycin A, 0,5 mM TMPD (N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride, Santa Cruz Biotechnology) + 1millimolar (mM) ascorbic acid, and 50millimolar (mM) azide.
Following each injection, record three measurements for a total period of 0h 15m 0s .
Calculate Complex I-, II-, and IV-dependent respiration by subtracting OCR values from the substrates (Pyruvate + malate + ADP for CI, Succinate + rotenone for CII and TMPD + ascorbic acid for CIV) subtracted from the ones from the inhibitors (rotenone for CI, antimycin A + rotenone for CII and azide for CIV).
Normalize the experimental values to the protein content per well via a BCA assay.
