Isolation of Mouse Lymphocytes from Small Intestine Tissues

Brian S. Sheridan1, Leo Lefrançois1

1 University of Connecticut Health Center, Farmington, Connecticut
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 3.19
DOI:  10.1002/0471142735.im0319s99
Online Posting Date:  November, 2012
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Abstract

The isolation of lymphocytes and other hematopoietic‐derived cells from small intestinal tissues has become increasingly relevant to immunology over the last decade. It is also becoming increasingly clear that the impact of local immunity at the mucosal barrier of the intestine has a profound impact on immune responses at distant sites, bringing a new cadre of immunologists to the mucosal frontier. Furthermore, the ability to experimentally manipulate smaller and smaller populations of immune cells has become technologically feasible and in some cases routine. The expanding importance of mucosal immunology coupled with increased technical capabilities requires a standard for experimentally obtaining uniform and consistent cells from the intestinal mucosa. Therefore, it is important to isolate immune cells that are highly viable and minimally manipulated to maximize cellular yields while maintaining acceptable time constraints. Curr. Protoc. Immunol. 99:3.19.1‐3.19.11. © 2012 by John Wiley & Sons, Inc.

Keywords: lymphocyte; small intestine; intraepithelial lymphocyte; lamina propria; Peyer's patches

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Intestinal Intraepithelial Lymphocytes
  • Basic Protocol 2: Isolation of Peyer's Patch Immune Cells
  • Basic Protocol 3: Isolation of Intestinal Lamina Propria Lymphocytes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Intestinal Intraepithelial Lymphocytes

  Materials
  • Mice: e.g., strain C57Bl/6, 10 weeks old
  • CMF solution (see recipe), 4°C
  • DTE solution (see recipe), 37°C
  • Harvest medium (see recipe), 4°C
  • 44% and 67% Percoll solutions (see recipe), ambient temperature
  • Fetal bovine serum (FBS), heat‐inactivated ( appendix 2A)
  • Iris scissors (1 straight and 1 curved)
  • Small forceps, sharp, thin, and curved at the tip
  • 20‐ml syringes
  • 18‐ or 20‐G gavage needles
  • 50‐ and 15‐ml conical tubes
  • Vortex mixer
  • 70‐µm cell strainers
  • Refrigerated centrifuge capable of centrifugation of 50‐ and 15‐ml conical tubes up to 600 × g
  • 17 × 100–mm round‐bottom polystyrene tubes
  • Pipetting needle
  • 2.5‐ml plastic transfer pipets
  • Pasteur pipets
  • Dissecting scope or lighted magnifying glass
  • 50‐ml Erlenmeyer flasks, silanized (see recipe or appendix 3K)
  • Stir bars
  • Magnetic stir plate
  • Thermostat‐controlled incubator large enough to contain stir plate, 37°C
  • Additional reagents and equipment for euthanasia (unit 1.8) and trypan blue exclusion and cell counting ( appendix 3B)

Basic Protocol 2: Isolation of Peyer's Patch Immune Cells

  Materials
  • Collagenase solution (see recipe), 37°C
  • Harvest medium (see recipe), 4°C
  • 15‐ml conical tubes
  • Thermostat controlled shaking incubator, 37°C
  • Vortex mixer
  • 70‐µm cell strainers
  • 3‐ml syringes
  • Refrigerated centrifuge capable of centrifugation of 15‐ml conical tubes up to 400 × g
  • Additional reagents and equipment for trypan blue dye exclusion ( appendix 3B) and cell counting

Basic Protocol 3: Isolation of Intestinal Lamina Propria Lymphocytes

  Materials
  • EDTA solution (see recipe), 37°C
  • Harvest medium (see recipe), 4°C and 37°C
  • Collagenase solution (see recipe), 37°C
  • 44% and 67% Percoll solutions (see recipe), ambient temperature
  • Fetal bovine serum (FBS), heat‐inactivated ( appendix 2A)
  • 50‐ml Erlenmeyer flasks, silanized (see recipe or appendix 3K)
  • Stir bars
  • Magnetic stir plate
  • Thermostat‐controlled incubator large enough to contain stir plate, 37°C
  • 70‐µm cell strainers
  • 3‐ml syringes
  • Refrigerated centrifuge capable of centrifugation of 50‐ml conical tubes up to 600 × g
  • 17 × 100–mm round‐bottom polystyrene tubes
  • Pipetting needle
  • Pasteur pipet
  • Additional reagents and equipment for trypan blue dye exclusion ( appendix 3B) and cell counting
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Figures

Videos

Literature Cited

Literature Cited
   Lefrançois, L. 1987. Carbohydrate differentiation antigens of murine T cells: Expression on intestinal lymphocytes and intestinal epithelium. J. Immunol. 138:3375‐3384.
   Lefrançois, L. 1991. Phenotypic complexity of intraepithelial lymphocytes of the small intestine. J. Immunol. 147:1746‐1751.
   Mowat, A.M. 2003. Anatomical basis of tolerance and immunity to intestinal antigens. Nat. Rev. Immunol. 3:331‐341.
   Sheridan, B.S. and Lefrancois, L. 2010. Intraepithelial lymphocytes: To serve and protect. Curr. Gastroenterol. Rep. 12:513‐521.
   Sheridan, B.S. and Lefrancois, L. 2011. Regional and mucosal memory T cells. Nat. Immunol. 12:485‐491.
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