High Throughput‐Based Mitochondrial Function Assays by Multi‐Parametric Flow Cytometry

J. Paul Robinson1, Nianyu Li2, Padma Kumar Narayanan2

1 Purdue University Cytometry Laboratories, West Lafayette, Indiana, 2 Amgen, Thousand Oaks, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.48
DOI:  10.1002/0471142956.cy0948s73
Online Posting Date:  July, 2015
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Abstract

Mitochondrial dysfunction has been increasingly implicated as an important mechanism for chemical‐induced toxicity. In the present unit, we describe a multi‐parametric flow cytometry assay to assess the effects of drug or chemical‐induced mitochondrial dysfunction in cells. Cells are cultured in a glucose‐supplemented medium and exposed to increasing concentrations of various chemicals. Several key mitochondrial/cellular parameters known to be directly impacted by mitochondrial dysfunction, such as mitochondrial membrane potential (MMP), mitochondrial reactive oxygen species (ROS) production, intracellular reduced glutathione (GSH) level, and cell viability, are simultaneously measured by flow cytometry. © 2015 by John Wiley & Sons, Inc.

Keywords: mitochondria; flow cytometry; mitochondrial dysfunction; mitochondrial membrane potential; reactive oxygen species (ROS); cell viability

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Multi‐Function Assay
  • Support Protocol 1: Automated 384‐Well‐Based High‐Throughput Flow Cytometry Assay
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Multi‐Function Assay

  Materials
  • HL‐60 cells or other appropriate cell lines in suspension
  • Complete cell culture medium (e.g., RPMI‐1640 with 10% heat‐inactivated FBS, 2 mM L‐glutamine, and 10 U penicillin/streptomycin for HL‐60 cells)
  • Antimycin A (Sigma‐Aldrich, cat. no. A8674)
  • Dimethyl sulfoxide (DMSO)
  • Fluorescent probes:
    • Calcein‐AM (Invitrogen, cat. no. C3099)
    • JC‐1 (Invitrogen, cat. no. T3168)
    • Monobromobimane (mBBr; Invitrogen, cat. no. M1378)
    • MitoSOX Red mitochondrial superoxide indicator (MitoSOX; Invitrogen, cat. no. M36008)
  • 96‐well round‐bottom tissue culture‐treated plates

Support Protocol 1: Automated 384‐Well‐Based High‐Throughput Flow Cytometry Assay

  Additional Materials (also see protocol 1Basic Protocol)
  • 384‐well round‐bottom tissue culture‐treated plates
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Figures

Videos

Literature Cited

Literature Cited
  Cossarizza, A., Baccarani‐Contri, M., Kalashnikova, G., and Franceschi, C. 1993. A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J‐aggregate forming lipophilic cation 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimidazolcarbocyanine iodide (JC‐1). Biochem. Biophys. Res. Commun. 197:40‐45.
  DiMauro, S. and Schon, E.A. 2003. Mitochondrial respiratory‐chain diseases. N. Engl. J. Med. 348:2656‐2668.
  Dykens, J.A. and Will, Y. 2007. The significance of mitochondrial toxicity testing in drug development. Drug Discov. Today 12:777‐785.
  Grieshaber, P., Lagreze, W.A., Noack, C., Boehringer, D., and Biermann, J. 2010. Staining of fluorogold‐prelabeled retinal ganglion cells with calcein‐AM: A new method for assessing cell vitality. J. Neurosci. Methods 192:233‐239.
  Hedley, D., and Chow, S. 1994. Glutathione and cellular resistance to anti‐cancer drugs. Methods Cell Biol. 42 Pt B:31‐44.
  Hedley, D., and Chow, S. 1994. Glutathione and cellular resistance to anti‐cancer drugs. In Methods in Cell Biology, 2nd edition (Z. Darzynkiewicz, J.P. Robinson, and H.A. Crissman, eds.) pp. 31‐44. Academic Press, San Diego.
  Krutzik, P.O. and Nolan, G.P. 2006. Fluorescent cell barcoding in flow cytometry allows high‐throughput drug screening and signaling profiling. Nat. Methods 3:361‐368.
  Li, N., Oquendo, E., Capaldi, R.A., Robinson, J.P., He, Y.D., Hamadeh, H.K., Afshari, C.A., Lightfoot‐Dunn, R., and Narayanan, P.K. 2014. A systematic assessment of mitochondrial function identified novel signatures for drug‐induced mitochondrial disruption in cells. Toxicol. Sci. 142:261‐273.
  Marroquin, L.D., Hynes, J., Dykens, J.A., Jamieson, J.D., and Will, Y. 2007. Circumventing the Crabtree Effect: Replacing media glucose with galactose increases susceptibility of HepG2 cells to mitochondrial toxicants. Toxicol. Sci. 97:539‐547.
  Mukhopadhyay, P., Rajesh, M., Yoshihiro, K., Hasko, G., and Pacher, P. 2007. Simple quantitative detection of mitochondrial superoxide production in live cells. Biochem. Biophys. Res. Commun. 358:203‐208.
  Pereira, C.V., Moreira, A.C., Pereira, S.P., Machado, N.G., Carvalho, F.S., Sardao, V.A., and Oliveira, P.J. 2009. Investigating drug‐induced mitochondrial toxicity: A biosensor to increase drug safety? Curr. Drug Saf. 4:34‐54.
  Robinson, J.P., Patsekin, V., Holdman, C., Ragheb, K., Sturgis, J., Fatig, R., Avramova, L.V., Rajwa, B., Davisson, V.J., Lewis, N., Narayanan, P., Li, N., and Qualls, C.W. Jr. 2013. High‐throughput secondary screening at the single‐cell level. J. Lab. Autom. 18:85‐98.
  Sklar, L.S., Carter, M.B., and Edwards, B.S. 2007. Flow cytometry for drug discovery, receptor pharmacology and high‐throughput screening. Curr. Opin. Pharmacol. 7:527‐534.
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