Assessing Mitochondrial Redox Status by Flow Cytometric Methods: Vascular Response to Fluid Shear Stress

Rongsong Li1, Nelson Jen1, Fei Yu1, Tzung K. Hsiai1

1 Cardiovascular Engineering Research, Department of Biomedical Engineering and Cardiology, School of Medicine and School of Engineering, University of Southern California, Los Angeles, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.37
DOI:  10.1002/0471142956.cy0937s58
Online Posting Date:  October, 2011
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Mitochondria are an important source of superoxide production contributing to physiological and pathological responses, including vascular oxidative stress that is relevant to cardiovascular diseases. Vascular oxidative stress is intimately linked with pro‐inflammatory states and atherosclerosis. Oxidized low‐density lipoprotein (OxLDL) modulates intracellular redox status and induces apoptosis in endothelial cells. Hemodynamic, specifically, fluid shear stress imparts both biomechanical and metabolic effects on vasculature. Mitochondria are an important source of superoxide production contributing to vascular oxidative stress with relevance to cardiovascular diseases. We hereby present biophysical and biochemical approaches, including fluorescence‐activated cell sorting, to assess the dynamics of vascular redox status. Curr. Protoc. Cytom. 58:9.37.1‐9.37.14. © 2011 by John Wiley & Sons, Inc.

Keywords: flow cytometry; oxidative stress; shear stress; mitochondrial redox status

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Table of Contents

  • Introduction
  • Basic Protocol 1: Assessing Mitohcondrrial Redox Status in Response to Fluid Shear Stress
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Assessing Mitohcondrrial Redox Status in Response to Fluid Shear Stress

  • Bovine (BAEC) or human aortic endothelial cells (HAEC)
  • High glucose (4.5 g/liter) DMEM (Invitrogen)
  • Heat‐inactivated fetal bovine serum (Hyclone)
  • 100 U/ml L‐glutamine‐penicillin‐streptomycin (Sigma)
  • JNK inhibitor SP600125 (10 µM) or NADPH oxidase inhibitor apocynin (1 mM) or anti‐oxidant N‐acetyl cysteine (NAC, 5 mM; Sigma)
  • siRNA target sequence for bovine JNK1: 5′‐CATGGAGCTCATGGATGCAAA‐TCTT‐3′ (30 nM)
  • siRNA target sequence for bovine JNK2: 5′‐CATGAAAGAATGTCCTACCTTCTTT‐3′. (30 nM)
  • Negative control siRNA (Qiagen)
  • Lipofectamine RNAiMAX (Invitrogen)
  • Rotenone (Sigma)
  • Oligomycin (Alexora)
  • FCCP [carbonylcyanide‐4‐(trifluoromethoxy)‐phenylhydrazone; Sigma]
  • Cyclosporin‐A (Sigma)
  • MnTMPyP (Alexora)
  • Cationic fluorescent dye, tetramethylrhodamine methyl ester (TMRM+; Molecular Probes)
  • Dulbecco's phosphate‐buffered saline (DPBS; Invitrogen)
  • Trypsin/EDTA (Invitrogen)
  • MitoSOX Red (Invitrogen)
  • Dulbecco's phosphate‐buffered saline supplemented with 2% FBS (Hyclone)
  • 2% Paraformaldehyde
  • Glass slides (5 cm2; BD Bioscience)
  • 37°C, 5% CO 2 incubator
  • FACS Caliber system (BD Biosciences)
  • Flow cytometer (e.g., BD LSR II, BD Biosciences)
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Literature Cited

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