Sep 23, 2023

Public workspaceMitochondrial isolation protocol

DOI
dx.doi.org/10.17504/protocols.io.kqdg3x4zzg25/v1
  • 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna (AT)
Elias Adriaenssens
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DOI: dx.doi.org/10.17504/protocols.io.kqdg3x4zzg25/v1
Protocol Citation: Elias Adriaenssens 2023. Mitochondrial isolation protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3x4zzg25/v1
Manuscript citation:
The methodology is based on Wieckowski et al. 2009 Nat Protocols.
Wieckowski, M., Giorgi, C., Lebiedzinska, M.et al. Isolation of mitochondria associated membranes and mitochondria from animal tissues and cells. Nat Protoc 4, 1582–1590 (2009).
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: June 26, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 84020
Keywords: Mitochondrial isolation, HeLa cells, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
Marie Skłodowska-Curie MSCA Postdoctoral fellowship
Grant ID: 101062916
Abstract
This protocol describes how to isolate crude mitochondrial fractions from HeLa cells.
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Materials
MATERIALS

Mitochondrial isolation buffer:
AB
HEPES (pH 7.4)5 mM
mannitol250 mM
EGTA0.5 mM
make fresh on the day itself!
Oligomycin/Antimycin A cocktail:
AB
Oligomycin10 µM
Antimycin A4 µM
In case cells were treated for more than 8 hours, we also added 10 µM Q-VD-OPh (A1901, ApexBio) to suppress apoptosis.
RIPA buffer:
AB
Tris-HCl pH 8.050 mM
NaCl150 mM
sodium deoxycholate0.50%
SDS0.10%
NP-401%
supplemented by cOmplete EDTA-free protease inhibitors (11836170001, Roche) and phosphatase inhibitors (Phospho-STOP, 4906837001, Roche).
ReagentOligomycin AApexBio TechnologyCatalog #A5588

ReagentAntimycin A from Streptomyces sp.Merck MilliporeSigma (Sigma-Aldrich)Catalog #A8674

ReagentQ-VD-OPhApexBio TechnologyCatalog #A1901

ReagentcOmplete™, Mini, EDTA-free (Protease Inhibitor)RocheCatalog ##11836170001)

ReagentRoche PhosSTOP™Merck MilliporeSigma (Sigma-Aldrich)Catalog #4906837001

Mitochondrial isolation
Mitochondrial isolation
1h 15m
1h 15m
Seed HeLa cells in 15 cm dishes and grow until confluence. Treat the cells with DMSO or the mitophagy-inducing cocktail Oligomycin/Antimycin A (O/A) if needed.
Collect HeLa cells by trypsinization and resuspension in DMEM medium. Centrifuge the cells at Centrifigation300 x g, 4°C, 00:10:00 .
10m
Centrifigation
Resuspend the cell pellet in Amount1 mL of ice-cold PBS (1x) and spin again for Centrifigation300 x g, 4°C, 00:05:00 .
5m
Remove the PBS and resuspend the cell pellet in Amount1 mL mitochondrial isolation buffer, which was cooled to Temperature4 °C .
Note
Note that the mitochondrial isolation buffer is prepared fresh on the day itself.

To lyse cells without damaging the mitochondria, pipet the Amount1 mL cell suspension up and down (15-20x) with a 26.5G needle.
Note
This should lyse the plasma membrane but leave organelles intact.


Spin the suspension down at Centrifigation600 x g, 4°C, 00:10:00 .
10m
Centrifigation
Keep the supernatant and centrifugation Centrifigation600 x g, 4°C, 00:10:00 .
Note
The mitochondria are located in the supernatant at this speed, the pellet contains intact cells, cell nuclei, and other large cell debris.


10m
After two spins at Centrifigation600 x g , subject the supernatant to Centrifigation7000 x g, 4°C, 00:10:00 .

10m
Centrifigation
The pellet contains the mitochondria, so remove the supernatant and resuspend the pellet in Amount1 mL mitochondrial isolation buffer.
Then, centrifugation Centrifigation7000 x g, 4°C, 00:10:00 .
Note
The supernatant from the first spin at Centrifigation7000 x g can be stored as a cytosolic fraction.

10m
After two spins at Centrifigation7000 x g , subject the resuspended pellet to Centrifigation10000 x g, 4°C, 00:10:00 .
10m
Centrifigation
The pellet contains the mitochondria, so remove the supernatant and resuspend the pellet in Amount1 mL mitochondrial isolation buffer.
Then, centrifugation Centrifigation10000 x g, 4°C, 00:10:00 .
10m
After two spins at Centrifigation10000 x g , the pellet can be resuspended in RIPA lysis buffer or used for further procedures such as mitochondrial import assays, proteinase K protection assays, etc.