Mar 27, 2023

Public workspaceMitochondrial complex activity assays

DOI
dx.doi.org/10.17504/protocols.io.eq2ly753elx9/v1
  • 1German Center for Neurodegenerative Diseases (DZNE), Tübingen, 72076 Germany
Federico Bertoli
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DOI: dx.doi.org/10.17504/protocols.io.eq2ly753elx9/v1
Protocol Citation: michela.deleidi, María José Pérez J., Hariam Raji, Pascale Baden, Federico Bertoli 2023. Mitochondrial complex activity assays. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly753elx9/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 17, 2023
Last Modified: March 27, 2023
Protocol Integer ID: 78971
Keywords: Mitochondrial complex activity assays, mitochondrial electron transport chain
Abstract
Mitochondria complex activity assays measure the activity levels of the different complexes of the mitochondrial electron transport chain (ETC).
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676- 1425.docx
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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

ReagentMitoCheck® Complex I Activity Assay KitCayman Chemical CompanyCatalog #700930

ReagentQproteome 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
Mitochondrial complex activity assays
35m
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 Duration00:15:00 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 Amount3 mg of purified fresh mitochondria in Amount200 µL of MAS buffer (Concentration70 millimolar (mM) sucrose, Concentration220 millimolar (mM) mannitol, Concentration5 millimolar (mM) KH2PO4, Concentration5 millimolar (mM) MgCl2, Concentration1 millimolar (mM) EGTA, Concentration2 millimolar (mM) HEPES Ph7.4 ) and seed in XFpSeahorse microplates.
Centrifuge the plate at Centrifigation2000 x g, 4°C, 00:05:00 .
5m
Centrifigation
Measure the OCR before and after the serial addition of pyruvate + malate (Concentration5 millimolar (mM) each) + ADP 3,5 mM or 1 mM Succinate + Concentration4 micromolar (µM) rotenone, Concentration4 micromolar (µM) rotenone + Concentration8 micromolar (µM) antimycin A, 0,5 mM TMPD (N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride, Santa Cruz Biotechnology) + Concentration1 millimolar (mM) ascorbic acid, and Concentration50 millimolar (mM) azide.
Pipetting
Following each injection, record three measurements for a total period of Duration00:15:00 .
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.