Mar 31, 2023

Mitochondrial complex activity assays

DOI
https://dx.doi.org/10.17504/protocols.io.14egn2oxyg5d/v1
  • 1German Center for Neurodegenerative Diseases (DZNE), Tübingen, 72076 Germany
Federico Bertoli
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DOI: https://dx.doi.org/10.17504/protocols.io.14egn2oxyg5d/v1
Protocol Citation: michela.deleidi , Maria Jose Perez J. 2023. Mitochondrial complex activity assays. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn2oxyg5d/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 28, 2023
Last Modified: June 01, 2024
Protocol  Integer ID: 79527
Keywords: Mitochondrial complex activity assays, mitochondrial electron transport chain, ASAPCRN, mitochondria complex activity assay, mitochondrial complex activity, mitochondrial electron transport chain, mitochondria, complex activity assay, activity levels of the different complex, activity level, different complex
Abstract
Mitochondria complex activity assays measure the activity levels of the different complexes of the mitochondrial electron transport chain (ETC).
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Materials
Materials

  • pyruvate
  • malate
  • ADP
  • Succinate
  • rotenone
  • antimycin A
  • TMPD (N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride, Santa Cruz Biotechnology)
  • ascorbic acid
  • azide
  • Qproteome Mitochondrial isolation kit

MitoCheck® Complex I Activity Assay KitCayman Chemical CompanyCatalog #700930

Qproteome Mitochondria Isolation KitQiagenCatalog #37612
MAS buffer
AB
Sucrose70 mM
Mannitol220 mM
KH2PO45 mM
MgCl25 mM
EGTA1 mM
HEPES pH 7.42 mM
Mitochondrial complex activity assays
35m
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 00:15:00 in the presence of ubiquinone and potassium cyanide to inhibit complex IV and prevent oxidation of ubiquinone.
Note
To assess CI, CII, and CIV function, we used a respirometry approach based on XFp Extracellular Flux Analysis and then proceed with steps 4-9.

15m
To this end, resuspend 3 mg of purified fresh mitochondria in 200 µL of MAS buffer (70 millimolar (mM) sucrose, 220 millimolar (mM) mannitol, 5 millimolar (mM) KH2PO4, 5 millimolar (mM) MgCl2, 1 millimolar (mM) EGTA, 2 millimolar (mM) HEPES 7.4 ) and seed in XFpSeahorse microplates.
Centrifuge the plate at 2000 x g, 4°C, 00:05:00 .
5m
Measure the OCR before and after the serial addition of pyruvate + malate (5 millimolar (mM) each) + ADP 3,5 mM or 1 mM Succinate + 4 micromolar (µM) rotenone, 4 micromolar (µM) rotenone + 8 micromolar (µM) antimycin A, 0,5 mM TMPD (N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride, Santa Cruz Biotechnology) + 1 millimolar (mM) ascorbic acid, and 50 millimolar (mM) azide.
Following each injection, record three measurements for a total period of 00:15:00 .
15m
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.