Experimental Autoimmune Encephalomyelitis in the Mouse

Stephen D. Miller1, William J. Karpus1, Todd Scott Davidson2

1 Northwestern University, Evanston, Illinois, 2 NIAID, NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 15.1
DOI:  10.1002/0471142735.im1501s88
Online Posting Date:  February, 2010
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Abstract

This unit details the materials and methods required for both active induction and adoptive transfer of experimental autoimmune encephalomyelitis (EAE) in the SJL mouse strain using intact proteins or peptides from the two major myelin proteins: proteolipid protein (PLP) and myelin basic protein (MBP). Detailed materials and methods required for the purification of both PLP and MBP are also described. A protocol for isolating CNS‐infiltrating lymphocytes in EAE mice is included. Modifications of the specified protocols may be necessary for efficient induction of active or adoptive EAE in other mouse strains. Curr. Protoc. Immunol. 88:15.1.1‐15.1.20. © 2010 by John Wiley & Sons, Inc.

Keywords: CFA; complete Freund's adjuvant; CNS; central nervous system; EAE; experimental autoimmune encephalomyelitis; MBP; myelin basic protein; MOG; myelin oligodendrocyte glycoprotein; PLP; proteolipid protein

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Active Induction of EAE with PLP and MBP Protein or Peptide
  • Alternate Protocol 1: Adoptive Induction of EAE with PLP‐, MBP‐, or MOG‐Specific Lymphocytes
  • Support Protocol 1: Purification of Proteolipid Protein
  • Support Protocol 2: Purification of Myelin Basic Protein
  • Support Protocol 3: Isolation of CNS‐Infiltrating Lymphocytes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Active Induction of EAE with PLP and MBP Protein or Peptide

  Materials
  • Incomplete Freund's adjuvant (IFA; Difco)
  • Mycobacterium tuberculosis H37Ra (killed and desiccated; Difco)
  • Solutions of MBP or PLP (see Support Protocols protocol 31 and protocol 42); PLP 139‐151, PLP 178‐191, MOG 92‐106, MBP 84‐104, or MOG 35‐55.
  • Female SJL or C57BL/6 mice, 5 to 8 weeks old (e.g., Jackson Labs or Harlan Labs)
  • Pertussis toxin (List Biologicals)
  • Carbol fuchsin solution (optional; see recipe)
  • 13‐ml polystyrene test tube
  • 18‐ and 25‐G needles (Becton Dickinson)
  • 1‐ml glass tuberculin syringe with Luer‐lok (e.g., VWR)
  • Oster small‐animal clipper (model A2)

Alternate Protocol 1: Adoptive Induction of EAE with PLP‐, MBP‐, or MOG‐Specific Lymphocytes

  • Mishell‐Dutton balanced salt solution (BSS; see recipe)
  • Complete DMEM‐5 ( appendix 2A)
  • Recombinant IL‐12 (R&D Systems)
  • 100‐mesh stainless steel screen (e.g., Fisher)
  • 50‐ml conical polypropylene centrifuge tubes
  • 37°C, 7.5% CO 2 tissue culture incubator
  • Additional reagents and equipment for active induction of EAE with PLP, MBP, or MOG (see protocol 1), euthanasia (unit 1.8), removal of lymphoid organs (unit 1.9), and trypan blue exclusion ( appendix 3B)

Support Protocol 1: Purification of Proteolipid Protein

  Materials
  • Bovine brain (Pel‐Freez)
  • Chloroform/methanol (CM; see recipe), 4°C
  • Methanol, 4°C
  • Acetone (Aldrich), 4°C
  • CM/acetic acid (see recipe)
  • Diethyl ether (Aldrich)
  • 2‐chloroethanol (Aldrich)
  • Stainless steel Waring blender
  • Whatman no. 1 filter paper (Whatman or Fisher)
  • Corex centrifuge bottles (Corning)
  • Buchner funnel (Fisher)
  • Separatory funnel
  • Nitrogen gas flow
  • Lipophilic Sephadex LH‐60‐120 packed in 1 m × 2.5 cm, Teflon‐fitted column (both from Pharmacia Biotech or Sigma), equilibrated with CM/acetic acid
  • Spectrapor 12‐ to 14‐kD‐MWCO dialysis tubing (Spectrum or Fisher)
  • BCA assay kit (Pierce)
  • Additional reagents and equipment for dialysis ( appendix 3H) and SDS‐PAGE (unit 8.4)

Support Protocol 2: Purification of Myelin Basic Protein

  Materials
  • Bovine brain or spinal cord (Pel‐Freez)
  • Chloroform/methanol (CM; see recipe), 4°C
  • 0.01 N HCl (Aldrich)
  • Saturated (NH 4) 2SO 4 (ammonium sulfate; unit 2.7)
  • 0.25 N NaOH
  • Buchner funnel (Fisher)
  • Stainless steel Waring blender
  • Whatman no. 90 and no. 4 filter papers (Whatman or Fisher)
  • Corex centrifuge bottles (Corning)
  • Spectrapor 12‐ to 14‐kDa‐MWCO dialysis tubing (Spectrum or Fisher)
  • Additional reagents and equipment for dialysis ( appendix 3H)

Support Protocol 3: Isolation of CNS‐Infiltrating Lymphocytes

  Materials
  • Mouse
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 50 mM 2‐mercaptoethanol (2‐ME)
  • Neural Tissue Dissociation Kit (Miltenyi Biotec, cat. no. 130‐092‐628)
  • 0.9 M sucrose in HBSS (see appendix 2A for HBSS)
  • 150‐mm petri dish
  • Surgical instruments:
    • Hemostats
    • Scissors
    • Forceps
    • Scalpel
  • 25‐G butterfly
  • 12‐cc syringe
  • 19‐G needle
  • 6‐cc syringe
  • 60‐mm petri dishes
  • 2‐ml pipets
  • 15‐ml conical centrifuge tubes
  • Refrigerated centrifuge
  • Additional reagents and equipment for euthanasia of the mouse by CO 2 asphyxiation (unit 1.8)
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Figures

Videos

Literature Cited

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