High‐Content Imaging Assays for Identifying Compounds that Generate Superoxide and Impair Mitochondrial Membrane Potential in Adherent Eukaryotic Cells

Puja Billis1, Yvonne Will1, Sashi Nadanaciva1

1 Compound Safety Prediction, Worldwide Medicinal Chemistry, Pfizer Inc, Groton, Connecticut
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 25.1
DOI:  10.1002/0471140856.tx2501s59
Online Posting Date:  February, 2014
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Abstract

Reactive oxygen species (ROS) are constantly produced in cells as a result of aerobic metabolism. When there is an excessive production of ROS and the cell's antioxidant defenses are overwhelmed, oxidative stress occurs. The superoxide anion is a type of ROS that is produced primarily in mitochondria but is also generated in other regions of the cell including peroxisomes, endoplasmic reticulum, plasma membrane, and cytosol. Here, a high‐content imaging assay using the dye dihydroethidium is described for identifying compounds that generate superoxide in eukaryotic cells. A high‐content imaging assay using the fluorescent dye tetramethylrhodamine methyl ester is also described to identify compounds that impair mitochondrial membrane potential in eukaryotic cells. The purpose of performing both assays is to identify compounds that (1) generate superoxide at lower concentrations than they impair mitochondrial membrane potential, (2) impair mitochondrial membrane potential at lower concentrations than they generate superoxide, (3) generate superoxide and impair mitochondrial function at similar concentrations, and (4) do not generate superoxide or impair mitochondrial membrane potential during the duration of the assays. Curr. Protoc. Toxicol. 59:25.1.1‐25.1.14. © 2014 by John Wiley & Sons, Inc.

Keywords: oxidative stress; superoxide; dihydroethidium; mitochondrial membrane potential; TMRM; high‐content imaging

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: High‐Content Imaging Assay for Identifying Compounds that Generate Superoxide
  • Basic Protocol 2: High‐Content Imaging Assay for Identifying Compounds that Impair Mitochondrial Membrane Potential
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: High‐Content Imaging Assay for Identifying Compounds that Generate Superoxide

  Materials
  • Cells to be tested (e.g., THLE‐2 cells, ATCC# CRL‐2706)
  • Phenol red–free cell culture medium
  • Test compounds
  • Pure dimethyl sulfoxide (DMSO, Sigma, cat. no. D2650)
  • Rotenone (Sigma, cat. no. R8875) or antimycin A (Sigma, cat. no. A8674): positive control for superoxide generation
  • Dihydroethidium (DHE, see recipe)
  • Pure dimethylformamide (DMF, VWR, cat. no. 9221‐01)
  • Hoechst 33342 trihydrochloride, trihydrate (10 mg/ml, 16.2 mM, see recipe)
  • Black‐walled, clear‐bottom, 96‐well plates (some cell types, e.g., THLE‐2, require collagen‐coated plates, Becton Dickinson, cat. no. 356700)
  • 37°C, 5% CO 2, 95% humidified incubator
  • 96‐well plates (for diluting test compounds)
  • Automated fluorescence microscopic imaging system (e.g., Thermo Fisher Scientific Cellomics ArrayScan VTI High‐Content Screening Reader)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.

Basic Protocol 2: High‐Content Imaging Assay for Identifying Compounds that Impair Mitochondrial Membrane Potential

  Materials
  • Cells to be tested (e.g., THLE‐2 cells, ATCC# CRL‐2706)
  • Cell culture medium
  • Test compounds
  • Pure dimethyl sulfoxide (DMSO, Sigma, cat. no. D2650)
  • Carbonyl cyanide 3‐chlorophenylhydrazone (CCCP, Sigma, cat. no. C2759)
  • Tetramethylrhodamine methyl ester perchlorate (TMRM, see recipe)
  • Hoechst 33342 trihydrochloride, trihydrate (see recipe)
  • Hank's balanced salt solution (HBSS, Lonza, cat. no. 10‐527F), 37°C
  • Black‐walled, clear‐bottom, 96‐well plates (some cell types, e.g., THLE‐2, require collagen‐coated plates, available from Becton Dickinson, cat. no. 356700)
  • 37°C, 5% CO 2, 95% humidified incubator
  • 96‐well plates (for diluting compounds)
  • Light microscope
  • Automated fluorescence microscopic imaging system (e.g., Thermo Fisher Scientific Cellomics ArrayScan VTI High‐Content Screening Reader)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.
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Figures

Videos

Literature Cited

Literature Cited
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