Inducible Nitric Oxide Synthase Expression

Qiao‐wen Xie1

1 Kenneth S. Warren Laboratories, Tarrytown, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 10.9
DOI:  10.1002/0471140856.tx1009s04
Online Posting Date:  May, 2001
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Abstract

This unit contains three protocols that can be used to determine iNOS expression in mouse macrophage‐like cells, RAW 264.7, by measuring end product, protein, and mRNA. A bacterial product, lipopolysaccharide (LPS), stimulates iNOS expression in RAW 264.7 cells.

     
 
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Table of Contents

  • Basic Protocol 1: Detection of iNOS Expression Based on Nitrite Accumulation
  • Basic Protocol 2: Confirming iNOS Expression at Protein Level by Immunoblot
  • Basic Protocol 3: Detection of iNOS Expression by Northern Blot Analysis
  • Support Protocol 1: 5′‐End Labeling of Oligonucleotide Probes
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Detection of iNOS Expression Based on Nitrite Accumulation

  Materials
  • RAW264.7 cells (ATCC #TIB 71)
  • Complete RPMI medium/10% FBS with antibiotics (Life Technologies; see recipe)
  • 100 µg/ml lipopolysaccharide (LPS) working solution (see recipe)
  • 10 mM sodium nitrite (see recipe)
  • Griess reagent working solution (see recipe)
  • 100‐mm‐diameter tissue culture dishes
  • Platform shaker
  • Ultrasonic bath (Lab‐Line Instruments)
  • 96‐well microtiter plate
  • Microplate reader
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 2: Confirming iNOS Expression at Protein Level by Immunoblot

  Materials
  • 10× PBS (see recipe or purchase from Life Technologies)
  • Lysis buffer (see recipe)
  • Bradford protein assay reagent kit (Bio‐Rad)
  • NuPAGE Tris‐Acetate SDS Buffer Kit (Novex) containing:
    • 4× sample buffer (also see recipe)
    • 20× running buffer (also see recipe)
    • 10× sample reducing agent
  • Precast NuPAGE 3% to 8% Tris‐acetate gel, 1.5‐mm‐thick, 10 wells (Novex)
  • Multicolored protein standards (NEN Life Science Products)
  • 20× NuPAGE transfer buffer (Novex; see recipe)
  • Blocking solution (see recipe)
  • PVDF or nitrocellulose membrane
  • PBST (see recipe)
  • Mouse monoclonal antibody 1E8‐B8 (iNOS‐specific; R&D Systems)
  • SuperSignal West Dura Mouse IgG Detection Kit (Pierce) containing:
  •  Horseradish peroxidase (HRP)‐conjugated anti‐mouse IgG
  •  Luminal/enhancer solution
  •  Stable peroxide solution
  • Cell scraper (Falcon)
  • 15‐ml centrifuge tube
  • XCell II Mini‐Cell (Novex)
  • XCell II Blot Module (Novex)
  • Polyvinylidene difluoride (PVDF) or nitrocellulose membrane: pore size, 0.2 µM
  • Whatman 3MM filter paper or equivalent
  • Kodak X‐Omat AR X‐ray film
  • Additional reagents and equipment for culturing cells and inducing iNOS expression (see protocol 1), SDS‐PAGE ( appendix 3A) and immunoblotting (unit 2.3)
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 3: Detection of iNOS Expression by Northern Blot Analysis

  Materials
  • TRI Reagent for RNA isolation (Molecular Research Center)
  • Chloroform
  • Isopropanol
  • 75% ethanol
  • DEPC‐treated H 2O (see recipe)
  • 1% (w/v) agarose gel with 2% (v/v) formaldehyde (see recipe)
  • 10× MOPS buffer (see recipe)
  • Loading buffer (see recipe), freshly prepared
  • Formamide
  • 100% glycerol
  • RNA Molecular Size Standard (Life Technologies)
  • 20× SSPE, pH 7.4 (Life Technologies; also see recipe)
  • 50× Denhardt solution (see recipe)
  • 10% (w/v) sodium dodecyl sulfate (SDS)
  • 10 mg/ml herring sperm DNA (Promega)
  • 20× SSC: 3.0 M NaCl/0.3 M sodium citrate, pH 7.0 (Life Technologies)
  • 50‐ml Oak Ridge centrifuge tubes with caps, polypropylene (Nalge)
  • Centrifuge (e.g., Sorvall with SS‐34 rotor or equivalent)
  • 55° to 60°C and 65° to 70°C water baths
  • Horizontal electrophoresis apparatus, with circulation (OWL Separation Systems)
  • UV lightbox
  • GeneScreenPlus nylon membrane (NEN Life Scientific Products)
  • Turboblotter Rapid Downward Transfer System (Schleicher & Schuell)
  • Stratalinker UV cross‐linker (Stratagene)
  • Heat‐sealable bags (Kapak)
  • Impulse sealer (American International Electric)
  • Additional reagents and equipment for culturing cells and inducing iNOS expression (see protocol 1), determining RNA concentration by spectrophotometry ( appendix 3A), and preparing oligonucleotide probes specific to mouse iNOS and β‐actin (see protocol 4
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: 5′‐End Labeling of Oligonucleotide Probes

  Materials
  • Oligonucleotide iNOS‐34: 5′‐TCT GTG CTG TCC CAG TGA GGA GCT GCG GGG AGC C‐3′
  • Mouse β‐actin oligonucleotide (Clontech)
  • 10× kinase buffer (Promega; also see recipe)
  • 10 mCi/ml [γ‐32P]ATP (6000 Ci/mmol; NEN Life Scientific Products)
  • 10 U/µl T4 polynucleotide kinase (Promega)
  • 65°C water bath
  • Quick‐Spin columns (TE) for radiolabeled DNA purification, Sephadex G‐25 (Boehringer Mannheim)
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Figures

Videos

Literature Cited

Literature Cited
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Key References
   Green et al., 1982. See .
   Describes in detail the reduction of nitrate to nitrite and the measurement of total nitrite in the sample by a stoichiometric diazotization reaction using the Griess reagent to form a purple azo product.
   Nathan, C. and Xie, Q.‐W. 1994. Regulation of biosynthesis of nitric oxide. J. Biol. Chem. 269:13725‐13728.
   Summarizes the induction and regulation of inducible nitric oxide synthase.
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