Induction of TNBS Colitis in Mice

Frank Scheiffele1, Ivan J. Fuss1

1 National Institute of Allergy and Infectious Disease, NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 15.19
DOI:  10.1002/0471142735.im1519s49
Online Posting Date:  August, 2002
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The animal model described in this unit is one of several that have been used to study the immunopathogenesis of inflammatory bowel disease. The model employs the use of 2,4,6‐trinitrobenzenesulfonic acid (TNBS), which induces severe colonic inflammation when administered intrarectally in SJL/J mice. The colitis which results from this procedure presents clinical and histopathological findings that resemble those seen in Crohn's disease. The unit describes the critical parameters needed for successful induction of TNBS‐colitis as well methods for monitoring and grading disease levels. A support protocol for isolating lamina propria mononuclear cells from mouse colons is also included.

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Table of Contents

  • Basic Protocol 1: Induction and Evaluation of TNBS Colitis in Mice
  • Support Protocol 1: Isolation of Mouse Mesenteric Lymph Node Cells
  • Support Protocol 2: Isolation of Lamina Propria Mononuclear Cells (LPMCs) from Mouse Colons
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Induction and Evaluation of TNBS Colitis in Mice

  • 2,4,6‐trinitrobenzenesulfonic acid (TNBS; also called picrylsulfonic acid; Sigma)
  • 45% to 50% ethanol
  • Male 5‐ to 6‐week‐old mice of known weight (SJL/J preferred; Table 15.19.1)
  • Inhalable anesthetic (e.g., methoxyflurane, isoflurane, halothane)
  • Surgical lubricant (e.g., Surgilube; E. Fougera)
  • 3.5‐French 38‐cm, polyurethane catheter (Sherwood Medical)
  • 1‐ml disposable syringe
  • Additional reagents and equipment for administering inhaled anesthesia to mice (unit 1.4), fixing tissue in 10% PBS‐buffered formalin, and hematoxylin and eosin (H & E) staining (unit 12.8), immunohistochemistry (unit 21.4) and isolation of mesenteric lymph node cells (MLNCs; see protocol 2) lamina propria mononuclear cells (LPMCs; see protocol 3) from mouse colons.
    Table 5.9.1   MaterialsMouse Strain Susceptibility to TNBS‐Induced Colitis

    Strain Haplotype Classification
    ASW/Sn H‐25 Intermediate
    Balb/c H‐2d Susceptible
    C 3H/HeOU   H‐2K Intermediate
    C57/B6 H‐2b Resistant
    C57/B10 H‐2b Resistant
    DBA/2 H‐2d Resistant
    SJL H‐25 Highly susceptible

Support Protocol 1: Isolation of Mouse Mesenteric Lymph Node Cells

  • 1 to 2 mice treated with TNBS (see protocol 1)
  • 69% ethanol in a squeeze bottle
  • Hanks' balanced salt solution (HBSS; appendix 2A) without Ca2+ and Mg2+
  • Surgical instruments, sterile: scissors, forceps, and scalpel or razor blade
  • 10‐cm petri dish
  • Syringe plunger
  • 40‐ and 100‐µm nylon cell strainer
  • 50‐ml conical tube
  • Additional reagents and equipment for counting cells ( appendix 3A)

Support Protocol 2: Isolation of Lamina Propria Mononuclear Cells (LPMCs) from Mouse Colons

  • Mice treated with TNBS (see protocol 1)
  • recipeHBSS/EDTA (see recipe)
  • Iscove's modified Dulbecco medium supplemented with 10% FBS (IMDM‐10; appendix 2A) and 50 µg/ml gentamicin
  • 4000 Mandl units (3 × 106 Wünsch units)/ml collagenase D (Roche) in HBSS
  • 1 mg/ml DNase I (Roche) in HBSS
  • 70% and 30% Percoll: 7 parts:3 parts or 3 parts:7 parts recipe100% Percoll (see recipe): 1 mM EDTA/PBS ( appendix 2A)
  • PBS ( appendix 2A), free of Ca2+ and Mg2+
  • 10% PBS buffered formalin (Fisher Scientific)
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Literature Cited

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