Aug 20, 2025

Mitochondrial isolation from cultured fibroblasts

DOI
https://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
EDTAMerck MilliporeSigma (Sigma-Aldrich)Catalog #ED-1KG
Deionized water
0.5 M EDTAFisher ScientificCatalog #50-983-251
Dislodge cultured fibroblasts with trypsin
Spin cell down at 1500 rpm, 00:05:00

Discard supernatant and keep pellet On ice

Resuspend pellet in 1 mL Mitochondria Isolation Buffer (MIB; 215 millimolar (mM) mannitol, 75 millimolar (mM) sucrose, 0.1% BSA, 20 millimolar (mM) HEPES, and 1 millimolar (mM) EGTA, adjusted to pH 7.2 with KOH)

Spin suspension at 400 rcf, 00:05:00

Discard supernatant
Resuspend pellet in 450 µL EDTAMerck MilliporeSigma (Sigma-Aldrich)Catalog #ED-1KG

Break cells with a Nitrogen cell disruptor at 1250 Bar for 00:10:00

Pass lysates through a 26-gauge syringe topped with 1.5 mL MIB by spinning at 1300 x g, 4°C, 00:03:00

Repeat Step 9
Transfer supernatant to a new tube and spin 1300 x g, 4°C, 00:10:00

Apply supernatant to Ficoll gradient (25 mL STE [8.55 g sucrose, 32 µL 0.5 M EDTAFisher ScientificCatalog #50-983-251 , 80 µL 1 M Tris, up to 25 mL with Deionized water ] and Ficoll stock [10 mL STE ,17.5 mL 20% Ficoll])

Spin at 3200 rpm, 00:30:00

Discard supernatant
Resuspend pellet in 1 mL MIB

Spin at 13000 rcf, 00:10:00

Discard supernatant
Resuspend pellet (purified mitochondria) in MIB