Aug 20, 2025

Public workspaceMitochondrial isolation from cultured fibroblasts

DOI
dx.doi.org/10.17504/protocols.io.81wgbwxr1gpk/v1
  • Ashley Seifert1,
  • Vekaria Hemendra2,
  • Ebenezer Aryee2
  • 1University of Kentucky;
  • 2Medical University of South Carolina
Ashley Seifert
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DOI: https://dx.doi.org/10.17504/protocols.io.81wgbwxr1gpk/v1
Protocol Citation: Ashley Seifert, Vekaria Hemendra, Ebenezer Aryee 2025. Mitochondrial isolation from cultured fibroblasts. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbwxr1gpk/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: August 17, 2025
Last Modified: August 20, 2025
Protocol Integer ID: 224833
Keywords: ASAPCRN, mitochondria, fibroblasts, mitochondrial isolation from cultured fibroblast, mitochondria from cultured fibroblast, mitochondrial isolation, mitochondria, cultured fibroblast, seifert lab
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-020495
NIH
Grant ID: R01AR070313
Abstract
This protocol describes the procedure used by the Seifert Lab to isolate mitochondria from cultured fibroblasts.
Protocol materials
ReagentEDTAMerck MilliporeSigma (Sigma-Aldrich)Catalog #ED-1KG
ReagentDeionized water
Reagent0.5 M EDTAFisher ScientificCatalog #50-983-251
Troubleshooting
Dislodge cultured fibroblasts with trypsin
Spin cell down at Centrifigation1500 rpm, 00:05:00

Discard supernatant and keep pellet TemperatureOn ice

Resuspend pellet in Amount1 mL Mitochondria Isolation Buffer (MIB; Concentration215 millimolar (mM) mannitol, Concentration75 millimolar (mM) sucrose, 0.1% BSA, Concentration20 millimolar (mM) HEPES, and Concentration1 millimolar (mM) EGTA, adjusted to pH 7.2 with KOH)

Spin suspension at Centrifigation400 rcf, 00:05:00

Discard supernatant
Resuspend pellet in Amount450 µL ReagentEDTAMerck MilliporeSigma (Sigma-Aldrich)Catalog #ED-1KG

Break cells with a Nitrogen cell disruptor at Pressure1250 Bar for Duration00:10:00

Pass lysates through a 26-gauge syringe topped with Amount1.5 mL MIB by spinning at Centrifigation1300 x g, 4°C, 00:03:00

Repeat Step 9
Transfer supernatant to a new tube and spin Centrifigation1300 x g, 4°C, 00:10:00

Apply supernatant to Ficoll gradient (Amount25 mL STE [Amount8.55 g sucrose, Amount32 µL Reagent0.5 M EDTAFisher ScientificCatalog #50-983-251 , Amount80 µL 1 M Tris, up to Amount25 mL with ReagentDeionized water ] and Ficoll stock [Amount10 mL STE ,Amount17.5 mL 20% Ficoll])

Spin at Centrifigation3200 rpm, 00:30:00

Discard supernatant
Resuspend pellet in Amount1 mL MIB

Spin at Centrifigation13000 rcf, 00:10:00

Discard supernatant
Resuspend pellet (purified mitochondria) in MIB