Single‐Cell Analysis of Lipid Rafts in Lymphocytes and in T Cell–Containing Immunoconjugates

William T. Lee1, Andrew R. O. Watson2

1 The Wadsworth Center, Albany, New York, 2 The University at Albany, Albany, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 2.11
DOI:  10.1002/0471140856.tx0211s27
Online Posting Date:  March, 2006
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Abstract

Within the plasma membranes of many different cell types, certain membrane lipids, including cholesterol and sphingolipids, form lateral assemblies surrounded by unsaturated glycerophospholipids. The concentration of such membrane lipids and associated proteins results in the formation of microdomains termed lipid rafts” (or glycolipid‐enriched membranes or detergent‐insoluble glycosphingolipid‐enriched domains). Proteins that possess saturated acyl chains are generally associated with lipid rafts. Lipid rafts are believed to be involved in a number of cellular processes including cell activation. When material is limiting, raft‐associated proteins may be identified on single cells using microscopy. This unit describes the application of this technique in an immunological example, examining the location and movement of signal transduction complexes in single T lymphocytes and in interactive conjugates between T cells and antigen‐presenting cells (APCs).

Keywords: plasma membrane; cholesterol; shingolipids; lipid rafts; T cell; antigen‐presenting cells

     
 
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Table of Contents

  • Basic Protocol 1: Identification of Lipid Rafts and Raft‐Associated Molecules in Single Cells
  • Basic Protocol 2: Identification of Lipid Rafts and Raft‐Associated Molecules During Cell‐Cell Interactions
  • Support Protocol 1: Preparation of Poly‐L‐Lysine Coated Slides
  • Support Protocol 2: Raft Disruption Using Methyl‐β‐Cyclodextrin (MβCD)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Identification of Lipid Rafts and Raft‐Associated Molecules in Single Cells

  Materials
  • Spleen or lymph nodes or CD4+ T cells or tissue culture cells
  • Complete RPMI 1640 (see recipe), 37°C
  • PBS/1% (w/v) BSA and PBS/0.1% (w/v) BSA
  • Cholera toxin B–rhodamine (CT‐B‐rhodamine; List Biological Laboratories) or cholera toxin B–Alexa Fluor 594 (Invitrogen Molecular Probes) at 10 µg/ml in PBS/0.1% BSA
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Anti‐CT‐B antibody (Calbiochem‐Novabiochem)
  • 4% (w/v) paraformaldehyde (see recipe)
  • 1% and 0.2% (v/v) Triton X‐100 extraction buffers (see recipe)
  • Pre‐titrated primary or directly conjugated fluorescent antibody
  • Pre‐titrated fluorescent secondary (detecting) antibody, optional
  • SlowFade Light Antifade Kit (Molecular Probes)
  • 15‐ml centrifuge tubes
  • 12‐well multitest slide (ICN) coated with poly‐L‐lysine (see protocol 3)
  • 37°C, 5% CO 2 humidified incubator
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper aseptic technique should be used accordingly.

Basic Protocol 2: Identification of Lipid Rafts and Raft‐Associated Molecules During Cell‐Cell Interactions

  Materials
  • Spleen or lymph nodes or CD4+ T cells and/or APCs or tissue culture cells
  • Complete RPMI 1640 (see recipe)
  • PBS/1% (w/v) BSA and PBS/0.1% (w/v) BSA
  • Cholera toxin B–rhodamine (CT‐B‐rhodamine; List Biological Laboratories) or cholera toxin B–Alexa Fluor 594 (Invitrogen Molecular Probes) at 10 µg/ml in PBS/0.1% BSA
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Anti‐CT‐B antibody (Calbiochem‐Novabiochem)
  • 4% (w/v) paraformaldehyde (see recipe)
  • 1% (v/v) Triton X‐100 and 0.2% (v/v) Triton X‐100 extraction buffers (see recipe)
  • SlowFade Light Antifade Kit (Molecular Probes)
  • 24‐well tissue culture plate
  • 15‐ml tubes
  • 1.5‐ml microcentrifuge tubes
  • 12‐well multitest slide (ICN) coated with poly‐L‐lysine (see protocol 3), 37°C
  • 37°C, 5% CO 2 humidified incubator
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper aseptic technique should be used accordingly.

Support Protocol 1: Preparation of Poly‐L‐Lysine Coated Slides

  Materials
  • 70% ethanol
  • 0.01% (w/v) poly‐L‐lysine solution: 0.1% commercial poly‐L‐lysine solution (Sigma) diluted 1:10 with deionized water
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 12‐well multiset slide (ICN)

Support Protocol 2: Raft Disruption Using Methyl‐β‐Cyclodextrin (MβCD)

  Materials
  • Cells
  • 10 mM methyl‐β‐cyclodextrin (MβCD; Sigma) dissolved in PBS
  • Phosphate‐buffered saline (PBS; appendix 2A)
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Figures

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Literature Cited

Literature Cited
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