Rapid isolation and purification of mitochondria for transplantation by tissue dissociation and differential filtration

Janine M. Preble, Hiroshi Kondo, James D. Mc Cully, Christina A. Pacak, Allison A. Mackay, Douglas B. Cowan

Research output: Contribution to journalArticlepeer-review

42 Scopus citations

Abstract

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator’s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 ± 0.04 g (wet weight) tissue sample.

Original languageEnglish (US)
Article numbere51682
JournalJournal of Visualized Experiments
Issue number91
DOIs
StatePublished - Jun 9 2014
Externally publishedYes

Bibliographical note

Publisher Copyright:
© JoVE 2006-2014. All Rights Reserved.

Keywords

  • Cellular Biology
  • Filtration
  • Homogenate
  • Investigative techniques
  • Isolation
  • Issue 91
  • Mitochondria
  • Operative
  • Surgical procedures
  • Tissue dissociation
  • Transplantation

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