Centrifugation‐Free Magnetic Isolation of Functional Mitochondria Using Paramagnetic Iron Oxide Nanoparticles

Bhabatosh Banik1, Shanta Dhar2

1 Department of Biochemistry and Molecular Biology, Miller School of Medicine, University of Miami, Miami, 2 Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 25.4
DOI:  10.1002/cpcb.26
Online Posting Date:  September, 2017
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Abstract

Subcellular fractionation techniques are essential for cell biology and drug development studies. The emergence of organelle‐targeted nanoparticle (NP) platforms necessitates the isolation of target organelles to study drug delivery and activity. Mitochondria‐targeted NPs have attracted the attention of researchers around the globe, since mitochondrial dysfunctions can cause a wide range of diseases. Conventional mitochondria isolation methods involve high‐speed centrifugation. The problem with high‐speed centrifugation‐based isolation of NP‐loaded mitochondria is that NPs can pellet even if they are not bound to mitochondria. We report development of a mitochondria‐targeted paramagnetic iron oxide nanoparticle, Mito‐magneto, that enables isolation of mitochondria under the influence of a magnetic field. Isolation of mitochondria using Mito‐magneto eliminates artifacts typically associated with centrifugation‐based isolation of NP‐loaded mitochondria, thus producing intact, pure, and respiration‐active mitochondria. © 2017 by John Wiley & Sons, Inc.

Keywords: mitochondria isolation; centrifugation‐free method; iron oxide nanoparticles; magnetic resonance imaging

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Mito‐Magneto
  • Basic Protocol 2: Isolation of Mitochondria from Cultured Cells
  • Support Protocol 1: Citrate Synthase Activity Assay
  • Support Protocol 2: Cytochrome C Oxidase Activity Assay
  • Support Protocol 3: Complex V Activity Assay
  • Support Protocol 4: ATP Production Assay
  • Support Protocol 5: Determination of Purity of Mitochondrial Fraction by Western Blotting
  • Support Protocol 6: TEM Imaging of Isolated Mitochondria
  • Alternate Protocol 1: Isolation of Mitochondria from Cultured Cells Using TOM22‐Based Magnetic Beads
  • Alternate Protocol 2: Isolation of Mitochondria from Cells Pretreated with Mitochondria‐Targeted and Non‐Targeted Polymeric Nanoparticles
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of Mito‐Magneto

  Materials
  • Tris(acetylacetonato)iron(III)—i.e., Fe(acac) 3
  • Benzyl ether
  • Oleic acid
  • Oleyl amine
  • 1,2‐hexadecanediol
  • Ethanol
  • Hexanes
  • 6‐bromohexanoic acid
  • Triphenylphosphine
  • Acetonitrile
  • Diethyl ether
  • Dimethyl formamide (DMF)
  • CDCl 3 (Cambridge Isotope Laboratories, cat. no. DLM‐7‐100)
  • DMSO‐d 6 (Cambridge Isotope Laboratories, cat. no. DLM‐10‐10)
  • H9C2 cells (a generous gift from Prof. Mark Anderson, University of Iowa)
  • 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (Sigma‐Aldrich, cat. no. M2128), for MTT assay
  • Dimethylsulfoxide (DMSO)
  • RAW 264.7 cells
  • Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 1% (w/v) L‐glutamine, 1% (w/v) sodium pyruvate, 1% penicillin‐streptomycin, and 10% (v/v) FBS (heat inactivated)
  • DMEM containing 1% DMSO
  • Lipopolysaccharide (LPS)
  • ELISA kits for IL‐6 (BD Biosciences, cat. no. 555240) and TNF‐alpha (BD Biosciences, cat. no. 558534)
  • Coating buffer (see recipe)
  • Wash buffer (see recipe)
  • Assay diluent (see recipe)
  • Stop solution (0.2 N sulfuric acid in water)
  • XF stress test optimization medium (DMEM with 1% penicillin/streptomycin) supplemented with sodium pyruvate, L‐glutamine, and D‐glucose
  • 2 μM oligomycin (Sigma‐Aldrich, cat. no. O4876‐25MG)
  • 2 μM FCCP (Sigma‐Aldrich, cat. no. C2920‐50MG)
  • 1 μM antimycin A (Sigma‐Aldrich, cat. no. A8674‐50MG) and rotenone (Sigma‐Aldrich, cat. no. R8875‐1G) mixture: combine equimolar amounts of antimycin A and rotenone, and dilute with XF stress test optimization medium to obtain a mixture containing 1 µM each
  • Sand bath (for heating reaction mixture at 200° to 300°C)
  • Nitrogen gas cylinder
  • Rotavap
  • Beckman Coulter Microcentrifuge 22R centrifuge
  • Branson 2510 sonicator bath
  • 1.5‐ml thick‐walled microcentrifuge tubes (Fisher Scientific, cat. no. 05‐408‐129)
  • 96‐well tissue culture plates (Sigma‐Aldrich, cat. no. SIAL‐0516)
  • 96‐well ELISA plates (Thermo Fisher Scientific, cat. no. 475094)
  • XF24 cell culture microplates (Agilent, cat. no. 102070‐001)
  • Additional reagents and equipment for Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM), Nuclear Magnetic Resonance (NMR), and Inductively Coupled Plasma Optical Emission Spectrometry (ICP‐OES), see Banik et al. ( )

Basic Protocol 2: Isolation of Mitochondria from Cultured Cells

  Materials
  • 20 µg/ml Mito‐magneto solution in medium containing 1% DMSO (from protocol 1)
  • 150‐cm2 cell culture flasks containing confluent monolayer (1–5 × 107 cells) of H9C2 cardiomyocytes, J3TBG glioma, or A2780 ovarian cancer cells
  • Cell culture media
    • For H9C2 and J3TBG cells: Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 1% (w/v) L‐glutamine, 1% (w/v) sodium pyruvate, 1% penicillin‐streptomycin, and 10% (v/v) FBS
    • For A2780 cells: Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% (v/v) FBS, 1% penicillin‐streptomycin, 1% (w/v) HEPES, and 2 mM L‐glutamine
  • Phosphate buffered saline (1× PBS), pH 7.4
  • 0.25% trypsin‐EDTA solution (Sigma‐Aldrich, cat. no. T4049)
  • Reagents A and C, ice cold, from mitochondria isolation kit (Thermo Fisher Scientific, cat. no. 89874)
  • 1× mitochondria assay solution (see recipe)
  • Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, cat. no. 23227)
  • 15‐ml conical tubes
  • BD Falcon 5‐ml round‐bottom tubes with cell strainer caps (BD Biosciences, cat. no. 352235)
  • Misonix probe sonicator (Newtown, CT)
  • EasySep magnet (Stemcell technologies, cat. no. 18000)
  • 1.5‐ml, thick‐walled microcentrifuge tubes
  • Malvern Zetasizer Nano ZS DLS system
  • Agilent (7 Tesla, 200 mm) horizontal bore magnet‐based MRI instrument

Support Protocol 1: Citrate Synthase Activity Assay

  Materials
  • Isolated mitochondria in 1× MAS (from protocol 2, steps 18a and 18b)
  • Citrate synthase activity assay kit (BioVision, cat. no. K318), containing citrate synthase assay buffer, citrate synthase substrate mix, citrate synthase developer, and glutathione (GSH) standards
  • 96‐well plates
  • Bio‐Tek Synergy HT microplate reader

Support Protocol 2: Cytochrome C Oxidase Activity Assay

  Materials (also see protocol 3)
  • COX activity kit (BioVision, cat. no. K287‐100), containing cytochrome C solution, positive control, and assay buffer

Support Protocol 3: Complex V Activity Assay

  Materials (also see protocol 3)
  • 5.5 mg/ml digitonin (Sigma‐Aldrich, cat. no. D141)
  • 1× MAS (see recipe)
  • 2 mM oligomycin solution
  • MitoTox Complex V OXPHOS Activity microplate assay kit (Abcam), consisting of complex V activity buffer, which contains ATP, pyruvate kinase (PK), lactate dehydrogenase (LDH), phosphoenolpyruvate, and NADH
  • Black‐bottomed, 96‐well plates (Thermo Fisher Scientific, cat. no. M33089)

Support Protocol 4: ATP Production Assay

  Materials (also see protocol 3)
  • 1 mM ADP solution
  • 1× MAS (see recipe)
  • CellTiter‐Glo luminescent cell viability assay kit (Promega, cat. no. G7574)

Support Protocol 5: Determination of Purity of Mitochondrial Fraction by Western Blotting

  Materials
  • Cellular fractions (nucleus and debris, cytosolic, and mitochondrial) suspended in 1× MAS (from protocol 2)
  • 4× Laemmli sample buffer (BioRad, cat. no. 161‐0747)
  • 10% (w/v) sodium dodecyl sulfate‐polyacrylamide (SDS‐PAGE) gel
  • Nitrocellulose membrane
  • Tris‐buffered saline with Tween 20 (TBST) buffer (see recipe)
  • Skimmed milk
  • Primary antibodies against TFAM (Abcam, cat. no. ab131607), VDAC1/porin (Abcam, cat. no. ab14737), lamin‐A (Abcam, cat. no. ab26300), and calnexin (Abcam, cat. no. ab10286)
  • HRP‐conjugated secondary goat anti‐rabbit IgG H&L (Abcam, cat. no. ab6721) and goat anti‐mouse IgG (Abcam, cat. no. ab97023) H&L antibodies, preadsorbed
  • Clarity western ECL substrate (BioRad, cat. no. 102030621 and 102030623)
  • FluorChem HD2 imaging system (Alpha Innotech), or similar
  • Orbital shaker

Support Protocol 6: TEM Imaging of Isolated Mitochondria

  Materials
  • 1× MAS (see recipe)
  • 2% (w/v) glutaraldehyde (Electron Microscopy Sciences, cat. no. 16000) in 1× MAS
  • 1% (w/v) OsO 4 (Electron Microscopy Sciences, cat. no. 19140) in 1× MAS
  • 30%, 50%, 75%, 95%, and 100% (v/v) ethanol in deionized water
  • Propylene oxide (Electron Microscopy Sciences, cat. no. 20401)
  • Spurr (Electron Microscopy Sciences, cat. no. 14300)
  • Araldi (Electron Microscopy Sciences, cat. no. 13940)
  • Copper grids (Electron Microscopy Sciences, cat. no. CF300‐Cu)
  • JEOL JEM 1011 Transmission Electron Microscope, or similar

Alternate Protocol 1: Isolation of Mitochondria from Cultured Cells Using TOM22‐Based Magnetic Beads

  Additional Materials (also see protocol 2)
  • Mitochondria MidiMACS Starting Kit (Miltenyi Biotec, cat. no. 130‐094‐872), containing lysis buffer, anti‐TOM22 microbeads, LS column, and magnet
  • Orbital shaker
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Figures

Videos

Literature Cited

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