title: Rapid isolation and purification of mitochondria for transplantation by tissue dissociation and differential filtration.
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 10(10) viable and respiration competent mitochondria from 0.18 ± 0.04 g (wet weight) tissue sample.
authors.affiliation: Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School.
authors.forename: Janine M
authors.initials: JM
authors.lastname: Preble
authors.affiliation: Department of Anesthesiology, Perioperative and Pain Medicine, Boston Children's Hospital and Department of Anesthesia, Harvard Medical School.
authors.forename: Christina A
authors.initials: CA
authors.lastname: Pacak
authors.affiliation: Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kondo
authors.affiliation: Department of Anesthesiology, Perioperative and Pain Medicine, Boston Children's Hospital and Department of Anesthesia, Harvard Medical School.
authors.forename: Allison A
authors.initials: AA
authors.lastname: MacKay
authors.affiliation: Department of Anesthesiology, Perioperative and Pain Medicine, Boston Children's Hospital and Department of Anesthesia, Harvard Medical School.
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Cowan
authors.affiliation: Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School; james_mccully@hms.harvard.edu.
authors.forename: James D
authors.initials: JD
authors.lastname: McCully
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D005374:Filtration / Q000295:instrumentation / Q000379:methods*; D008163:Luminescent Measurements / Q000379:methods*; D008928:Mitochondria / Q000737:chemistry* / Q000637:transplantation*; D009757:Nylons
chemical_list: D009757:Nylons; D000255:Adenosine Triphosphate