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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

Abstract

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential (MMP), mitochondrial production of reactive oxygen species (ROS), and mitochondrial DNA (mtDNA) copy number are often features of these processes. This report details a novel flow cytometry-based approach to measure multiple mitochondrial parameters in different cell types, including human induced pluripotent stem cells (iPSCs) and iPSC-derived neural and glial cells. This flow-based strategy uses live cells to measure mitochondrial volume, MMP, and ROS levels, as well as fixed cells to estimate components of the mitochondrial respiratory chain (MRC) and mtDNA-associated proteins such as mitochondrial transcription factor A (TFAM).

By co-staining with fluorescent reporters, including MitoTracker Green (MTG), tetramethylrhodamine ethyl ester (TMRE), and MitoSox Red, changes in mitochondrial volume, MMP, and mitochondrial ROS can be quantified and related to mitochondrial content. Double staining with antibodies against MRC complex subunits and translocase of outer mitochondrial membrane 20 (TOMM20) permits the assessment of MRC subunit expression. As the amount of TFAM is proportional to mtDNA copy number, the measurement of TFAM per TOMM20 gives an indirect measurement of mtDNA per mitochondrial volume. The entire protocol can be carried out within 2-3 h. Importantly, these protocols allow the measurement of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry.

Introduction

Mitochondria are essential organelles present in almost all eukaryotic cells. Mitochondria are responsible for energy supply by producing adenosine triphosphate (ATP) via oxidative phosphorylation and act as metabolic intermediaries for biosynthesis and metabolism. Mitochondria are deeply involved in many other important cellular processes, such as ROS generation, cell death, and intracellular Ca2+ regulation. Mitochondrial dysfunction has been associated with various neurodegenerative diseases, including Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), Friedreich's ataxia (FRDA), and amyotrophic lateral scleros....

Protocol

NOTE: See the Table of Materials and the Supplemental Table S1 for recipes of all media and solutions used in this protocol.

1. Differentiation of human iPSCs into NCSs, dopaminergic (DA) neurons, and astrocytes

  1. Prepare matrix-coated plates.
    1. Thaw a vial of 5 mL of matrix on ice overnight. Dilute 1 mL of matrix with 99 mL of cold Advanced Dulbecco's Modified Eagle Medium/Ham's F-12 (Advanced DMEM/F12) (1% final c.......

Representative Results

A schematic description of the differentiation method and flow cytometric strategies is shown in Figure 3. Human iPSCs are differentiated into neural rosettes and then lifted into suspension culture for differentiation into neural spheres. Neural spheres are further differentiated and matured into DA neurons. Neural spheres are dissociated into single cells to generate glial astrocytes, replated in monolayers as NSCs, and then differentiated into astrocytes. This protocol provides the strate.......

Discussion

Herein are protocols for generating iPSC−derived neurons and astrocytes and evaluating multiple aspects of mitochondrial function using flow cytometry. These protocols allow efficient conversion of human iPSCs into both neurons and glial astrocytes and the detailed characterization of mitochondrial function, mostly in living cells. The protocols also provide a co-staining flow cytometry-based strategy for acquiring and analyzing multiple mitochondrial functions, including volume, MMP, and mitochondrial ROS lev.......

Acknowledgements

We kindly thank the Molecular Imaging Centre and the Flow Cytometry Core Facility at the University of Bergen in Norway. This work was supported by funding from the Norwegian Research Council (Grant number: 229652), Rakel og Otto Kr.Bruuns legat and the China Scholarship Council (project number: 201906220275).

....

Materials

NameCompanyCatalog NumberComments
anti-Oct4Abcamab19857, RRID:AB_445175Primary Antibody; use as 1:100, 10 μL in 1000 μL staining solution; use Alexa Fluor ® 488 goat anti-rabbit IgG  (1:400, Thermo Fisher Scientific, Catalog # A-11008) as secondary antibody.
anti-SSEA4Abcamab16287, RRID:AB_778073Primary Antibody; use as 1:100, 10 μL in 1000 μL staining solution; use Alexa Fluor ® 594 goat anti-mouse IgG (1:800, Thermo Fisher Scientific, Catalog # A-11005) as secondary antibody.
anti-Sox2Abcamab97959, RRID:AB_2341193Primary Antibody; use as 1:100, 10 μL in 1000 μL staining solution; use Alexa Fluor ® 488 goat anti-rabbit IgG  (1:400, Thermo Fisher Scientific, Catalog # A-11008) as secondary antibody.
anti-Pax6Abcamab5790, RRID:AB_305110Primary Antibody; use as 1:100, 10 μL in 1000 μL staining solution; use Alexa Fluor ® 488 goat anti-rabbit IgG  (1:400, Thermo Fisher Scientific, Catalog # A-11008) as secondary antibody.
anti-NestinSanta Cruz Biotechnologysc-23927, RRID:AB_627994Primary Antibody; use as 1:50, 20 μL in 1000 μL staining solution; use Alexa Fluor ® 594 goat anti-mouse IgG (1:800, Thermo Fisher Scientific, Catalog # A-11005) as secondary antibody.
anti-GFAPAbcamab4674, RRID:AB_304558Primary Antibody; use as 1:100, 10 μL in 1000 μL staining solution;  use Alexa Fluor ® 594 goat anti-chicken IgG (1:800, Thermo Fisher Scientific, Catalog # A-11042) as secondary antibody.
anti-S100β  conjugated with Alexa Fluor 488Abcamab196442, RRID:AB_2722596Primary Antibody; use as 1:400, 2.5 μL in 1000 μL staining solution;
anti-THAbcamab75875, RRID:AB_1310786Primary Antibody; use as 1:100, 10 μL in 1000 μL staining solution; use Alexa Fluor ® 488 goat anti-rabbit IgG  (1:400, Thermo Fisher Scientific, Catalog # A-11008) as secondary antibody.
anti-Tuj 1Abcamab78078, RRID:AB_2256751Primary Antibody; use as 1:1000, 1 μL in 1000 μL staining solution; use Alexa Fluor ® 594 goat anti-mouse IgG (1:800, Thermo Fisher Scientific, Catalog # A-11005) as secondary antibody.
anti-SynaptophysinAbcamab32127, RRID:AB_2286949Primary Antibody; use as 1:100, 10 μL in 1000 μL staining solution; use Alexa Fluor ® 488 goat anti-rabbit IgG  (1:400, Thermo Fisher Scientific, Catalog # A-11008) as secondary antibody.
anti-PSD-95Abcamab2723, RRID:AB_303248Primary Antibody; use as 1:100, 10 μL in 1000 μL staining solution;  use Alexa Fluor ® 594 goat anti-chicken IgG (1:800, Thermo Fisher Scientific, Catalog # A-11042) as secondary antibody.
anti-TFAM conjugated with Alexa Fluor 488Abcamab198308Primary Antibody; use as 1:400, 2.5 μL in 1000 μL staining solution; use mouse monoclonal IgG2b  Alexa Fluor® 488 as an isotype control.
anti-TOMM20 conjugated with Alexa Fluor 488Santa Cruz BiotechnologyCat# sc-17764 RRID:AB_628381Primary Antibody; use as 1:400, 2.5 μL in 1000 μL staining solution; use mouse monoclonal IgG2a  Alexa Fluor® 488 as an isotype control.
anti-NDUFB10Abcamab196019Primary Antibody; use as 1:1000, 1 μL in 1000 μL staining solution; use Alexa Fluor ® 488 goat anti-rabbit IgG  (1:400, Thermo Fisher Scientific, Catalog # A-11008) as secondary antibody; use rabbit monoclonal IgG as an isotype control.
anti-SDHAAbcamab137040Primary Antibody; use as 1:1000, 1 μL in 1000 μL staining solution;  use Alexa Fluor ® 488 goat anti-rabbit IgG  (1:400, Thermo Fisher Scientific, Catalog # A-11008) as secondary antibody; use rabbit monoclonal IgG as an isotype control.
anti-COX IVAbcamab14744, RRID:AB_301443Primary Antibody; use as 1:1000, 1 μL in 1000 μL staining solution; use  Alexa Fluor ® 488 goat anti-mouse IgG  (1:400, Thermo Fisher Scientific, Catalog # A-11001) as secondary antibody; use mouse monoclonal IgG as an isotype control.
Activin APeproTech120-14EAstrocyte differentiation medium ingredient
ABM Basal MediumLonzaCC-3187Basal medium for astrocyte culture
AGM SingleQuots Supplement PackLonzaCC-4123Supplement for astrocyte culture
Antibiotic-AntimycoticThermo Fisher Scientific15240062CDM ingredient
Advanced DMEM/F-12Thermo Fisher Scientific12634010Basal medium for dilute Geltrex
Bovine Serum AlbuminEuropa BioproductsEQBAH62-1000Blocking agent to prevent non-specific binding of antibodies in immunostaining assays and CDM ingredient
BDNFPeproTech450-02DA neurons medium ingredient
B-27 SupplementThermo Fisher Scientific17504044Astrocyte differentiation medium ingredient
BD Accuri C6 Plus Flow CytometerBD Biosciences, USA
Chemically Defined Lipid ConcentrateThermo Fisher Scientific11905031CDM ingredient
Collagenase IVThermo Fisher Scientific17104019Reagent for gentle dissociation of human iPSCs
CCD Microscope Camera Leica DFC3000 GLeica Microsystems, Germany
Corning non-treated culture dishesSigma-AldrichCLS430589Suspension culture
DPBSThermo Fisher Scientific14190250Used for a variety of cell culture wash
DMEM/F-12, GlutaMAX supplementThermo Fisher Scientific10565018Astrocyte differentiation basal Medium
EDTAThermo Fisher Scientific15575020Reagent for gentle dissociation of human iPSCs
Essential 8 Basal MediumThermo Fisher ScientificA1516901Basal medium for iPSC culture
Essential 8 Supplement (50X)Thermo Fisher ScientificA1517101Supplement for iPSC culture
EGF Recombinant Human ProteinThermo Fisher ScientificPHG0314Supplement for NSC culture
FGF-basic (AA 10–155) Recombinant Human ProteinThermo Fisher ScientificPHG0024Supplement for NSC culture
Fetal Bovine SerumSigma-Aldrich12103CMedium ingredient
FGF-basicPeproTech100-18BAstrocyte differentiation medium ingredient
FCCPAbcamab120081Eliminates mitochondrial membrane potential and TMRE staining
Fluid aspiration system BVC controlVacuubrand, Germany
Formaldehyde (PFA) 16%Thermo Fisher Scientific28908Cell fixation
GeltrexThermo Fisher ScientificA1413302Used for attachment and maintenance of human iPSCs
GlutaMAX SupplementThermo Fisher Scientific35050061Supplement for NSC culture
GDNFPeprotech450-10DA neurons medium ingredient
GlycineSigma-AldrichG8898Used for blocking buffer
Ham's F-12 Nutrient MixThermo Fisher Scientific31765027Basal medium for CDM
Heregulin beta-1 humanSigma-AldrichSRP3055Astrocyte differentiation medium ingredient
Hoechst 33342Thermo Fisher ScientificH1399Stain the nuclei for confocal image
Heracell 150i CO2 IncubatorsFisher Scientific, USA
IMDMThermo Fisher Scientific21980032Basal medium for CDM
InsulinRoche1376497CDM ingredient
InSolution AMPK InhibitorSigma-Aldrich171261Neural induction medium ingredient
Insulin-like Growth Factor-I humanSigma-AldrichI3769Astrocyte differentiation medium ingredient
KnockOut DMEM/F-12 mediumThermo Fisher Scientific12660012Basal medium for NSC culture
LamininSigma-AldrichL2020Promotes attachment and growth of neural cells in vitro
Leica TCS SP8 STED confocal microscopeLeica Microsystems, Germany
MonothioglycerolSigma-AldrichM6145CDM ingredient
MitoTracker Green FMThermo Fisher ScientificM7514Used for mitochondrial volume indicator
MitoSox RedThermo Fisher ScientificM36008Used for mitochondrial ROS indicator
N-Acetyl-L-cysteineSigma-AldrichA7250Neural induction medium ingredient
N-2 SupplementThermo Fisher Scientific17502048Astrocyte differentiation medium ingredient
Normal goat serumThermo Fisher ScientificPCN5000Used for blocking buffer
Orbital shakers - SSM1Stuart Equipment, UK
Poly-L-ornithine solutionSigma-AldrichP4957Promotes attachment and growth of neural cells in vitro
Poly-D-lysine hydrobromideSigma-AldrichP7405Promotes attachment and growth of neural cells in vitro
PurmorphamineSTEMCELL Technologies72204Promotes DA neuron differentiation
ProLong Gold Antifade MountantThermo Fisher ScientificP36930Mounting the coverslip for confocal image
PBS 1xThermo Fisher Scientific18912014Used for a variety of wash
Recombinant Human/Mouse FGF-8b ProteinR&D Systems423-F8-025/CFPromotes DA neuron differentiation
SB 431542Tocris BioscienceTB1614-GMPNeural Induction Medium ingredient
StemPro Neural SupplementThermo Fisher ScientificA10508-01Supplement for NSCs culture
TrypLE Express EnzymeThermo Fisher Scientific12604013Cell dissociation reagent
TransferrinRoche652202CDM ingredient
TRITON X-100VWR International9002-93-1Used for cells permeabilization in immunostaining assays
TMREAbcamab113852Used for mitochondrial membrane potential staining
Water Bath Jb Academy Basic Jba5 JBA5 Grant InstrumentsGrant Instruments, USA

References

  1. Wang, Y., Xu, E., Musich, P. R., Lin, F. Mitochondrial dysfunction in neurodegenerative diseases and the potential countermeasure. CNS Neuroscience & Therapeutics. 25 (7), 816-824 (2019).
  2. Chen, A., et al.

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