Supported Planar Bilayers for Study of the Immunological Synapse

Michael L. Dustin1, Toby Starr1, Rajat Varma1, V. Kaye Thomas1

1 Skirball Institute of Biomolecular Medicine, New York, New York
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 18.13
DOI:  10.1002/0471142735.im1813s76
Online Posting Date:  February, 2007
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Abstract

Supported planar bilayers have been used in immunology research for over 25 years, including in the initial demonstrations of MHC‐peptide complex functional activity and adhesion molecule activity. More recent modifications of the method have been used to measure two‐dimensional affinities and to study the formation of the immunological synapse. This unit covers the incorporation of glycolipid‐anchored membrane proteins, 6‐histidine‐tagged soluble proteins, and monobiotinylated soluble proteins into supported planar bilayers. Reagents developed for the MHC‐peptide tetramer staining method (UNIT 17.3) can readily be adapted to presentation on planar bilayers. The unique advantage of this approach is that the proteins presented on the surface of the supported bilayer are laterally mobile. This provides a more physiological presentation of cell‐surface molecules and supports visualization of protein rearrangement on the bilayer by live cells.

Keywords: microscopy; receptors; affinity; fluorescence; signaling; activation

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Purification, Labeling, and Reconstitution of Glycosylphosphatidyl Inositol (GPI)–Anchored Proteins
  • Basic Protocol 2: Formation of Supported Bilayers on Glass Beads for Characterization by Flow Cytometry and in Flow Cell for Study of Immunological Synapse
  • Support Protocol 1: Preparation of Cleaned Glass Coverslips for Formation of Planar Bilayers
  • Basic Protocol 3: Immunological Synapse (IS) Formation
  • Support Protocol 2: Cleaning the Flow Cell
  • Support Protocol 3: Preparation of 5% Casein Blocking Reagent
  • Support Protocol 4: Preparation of T Cell Blasts
  • Alternate Protocol 1: Use of 6‐Histidine‐Tagged Proteins with Planar Bilayers
  • Alternate Protocol 2: Use of Biotinylated MHC Molecules with Planar Bilayers
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Purification, Labeling, and Reconstitution of Glycosylphosphatidyl Inositol (GPI)–Anchored Proteins

  Materials
  • >4 mg/ml solution of MAb (cell lines available from ATCC; see discussion above) in phosphate‐buffered saline (PBS; see recipe)
  • >4 mg/ml solution of irrelevant Ab (human IgG) in phosphate‐buffered saline (PBS; see recipe)
  • 4× coupling buffer (see recipe)
  • Cyanogen bromide–activated agarose beads (CNBr‐agarose, Sigma cat. no. C9210)
  • 1 mM HCl, cold
  • 100 mM ethanolamine, pH 8, or 0.2 M glycine, pH 8
  • Tris‐saline‐azide (TSA) buffer (see recipe), 4°C
  • CHO cells expressing I‐Ek‐GPI (M. Davis, Stanford University) and CD48 (P.A. van der Merwe, Oxford University) (see and introduction to this protocol)
  • BHK cells expressing ICAM‐1‐GPI and CD80‐GPI CD48 (Michael L. Dustin ) (see and introduction to this protocol)
  • DMEM containing 10% FBS ( appendix 2A)
  • HBSS ( appendix 2A) containing 5 mM EDTA and 1% (w/v) BSA
  • Liquid N 2 or dry ice/methanol bath
  • Lysis buffer (see recipe), with and without protease inhibitors
  • Tris‐saline buffer (see recipe)
  • Glycine elution buffer (see recipe)
  • Triethylamine elution buffer (see recipe)
  • Column wash buffer 1 (see recipe)
  • Labeling buffer (see recipe)
  • Reactive fluorescent dye (see recipe) for labeling: Cy5 (for ICAM‐1‐GPI) or Oregon Green 488 (for I‐Ek‐GPI)
  • Dimethylsulfoxide (DMSO; anhydrous; see recipe for reactive fluorescent dyes for further specifications)
  • Column wash buffer 2 (see recipe), ice cold
  • Column wash buffer 3 (see recipe), ice cold
  • Neutralization buffer:
    • 1 M Tris·Cl, pH 8.5 ( appendix 2A)/1% (w/v) octyl β‐D‐glucopyranoside (OG; Sigma) for low‐pH elution
    • 1 M Tris·Cl, pH 6.8 ( appendix 2A)/1% (w/v) octyl β‐D‐glucopyranoside (OG; Sigma) for high‐pH elution
  • 5‐µm diameter polystyrene beads (Polysciences) and FITC standard beads (Bangs Laboratories)
  • FACS buffer (see recipe)
  • FITC‐labeled MAb (or Fab) with known F/P ratio, of the same type used for purification
  • Nitrogen tank with regulator
  • Argon tank with regulator
  • Chloroform, high‐grade
  • 10 mg/ml dioleoylphosphatidylcholine (DOPC) in chloroform (see recipe for phospholipids)
  • 10% (w/v) octyl β‐D‐glucopyranoside (OG) in ultrapure H 2O (filter through 0.22‐µm filter; store at –20°C)
  • Ultracentrifuge with fixed‐angle rotor
  • Spectrophotometer
  • 60‐ml round‐bottom ultracentrifuge tubes (Beckman 355622)
  • 15‐, 30‐, and 50‐ml conical polypropylene centrifuge tubes
  • Sintered glass funnel, coarse, appropriately sized for amount of agarose to be washed
  • Side arm flask for vacuum filtration.
  • Spatula
  • End‐over‐end rotator
  • Refrigerated centrifuge with swinging‐bucket rotor
  • Cell Factories (Nunc)
  • 250‐ml conical plastic centrifuge bottles
  • Glass beaker of size appropriate for number of cells to be sonicated
  • Bronson Sonifier 450 probe sonicator with flat‐tip probe (or equivalent)
  • Millipore Stericup‐GP 150‐ or 250‐ml, 0.22‐µm filter units
  • 2‐ to 3‐ml plastic screw‐cap vials with O‐rings
  • Poly‐Prep disposable chromatography column (Bio‐Rad)
  • 96‐well, non–tissue culture treated V‐bottom microtiter plates
  • Plate‐sealing tape
  • Flow cytometer (e.g., Becton Dickinson FACSCalibur) and tubes
  • Platform shaker
  • 1‐liter wide‐mouth sterile medium bottles
  • 16 × 100–mm glass test tube
  • Bath sonicator
  • 0.22‐µm cellulose acetate filters (3‐mm Cameo 3AS; Thomas Scientific)
  • 1.5‐ml conical ultracentrifuge tubes (Beckman)
  • Dialysis tubing (Spectrum Spectra/Por 2 RC dialysis membrane, MWCO 12,000 to 14,000; see appendix 3H): 10 mm (for small proteoliposome volumes) or 25 mm flat width (for larger volumes of plain DOPC liposomes, NTA‐liposomes, or CAP‐biotin liposomes)
  • Dialysis tubing closures (see appendix 3H)
  • Boiling water bath
  • Additional reagents and equipment for protein assay (unit 2.11), SDS‐PAGE (unit 8.4), silver staining (unit 8.9), flow cytometry (Chapter 5), and dialysis ( appendix 3H)
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and proper aseptic technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO 2 to maintain pH 7.4.

Basic Protocol 2: Formation of Supported Bilayers on Glass Beads for Characterization by Flow Cytometry and in Flow Cell for Study of Immunological Synapse

  Materials
  • 0.4 mM dioleoylphosphatidylcholine (DOPC) liposomes in Tris‐saline (see protocol 1)
  • 0.4 mM proteoliposome preparation in Tris‐saline (see protocol 1)
  • 5‐µm silica beads (Bangs Laboratories, cat. no. SS05N; 9% solids/water suspension)
  • FACS buffer (see recipe)
  • FITC‐labeled antibody of known F/P molar ratio
  • FITC standard beads: Bangs Quantum low‐level (824p) and high‐level (825p), premixed
  • HBS/HSA buffer (see recipe)
  • 5% casein blocking reagent ( protocol 6)
  • Argon tank with regulator
  • 96‐well V‐bottom microtiter plate
  • Centrifuge with microtiter plate carrier
  • Microtiter plate shaker
  • Flow cytometer and tubes
  • Cleaned 40‐mm glass coverslips ( protocol 3)
  • Small screw‐cap vials
  • FCS2 flow cells with 0.25‐mm‐thickness rectangular gaskets (Bioptechs; Fig. )
  • 20‐ml and 1‐ml syringes
  • Three‐way and two‐way polypropylene valves (stopcocks)
  • Tygon tubing, R‐3603; i.d. 1/16 in., o.d. 1/8 in.
  • Fittings kit (Bio‐Rad)
  • FCS2 live‐cell micro‐observation system (Bioptechs)
  • Inverted microscope total internal reflection fluorescence
  • Microscope filters:
    • Dichroic mirror z488‐568‐647rpc (Chroma Technologies): designed to work with the authors' total internal reflection fluorescence microscopy (TIRFM) system with laser lines from Kr‐Ar laser 488, 568, and 647 nm; this element is fixed during experiments and emission filter wheels are changed to detect different fluorescent dyes
    • Single‐band excitation filters from Omega Optical (selected to work with the authors' 150‐W xenon arc lamp): XF1042 (485DF15) for Oregon Green; XF1045 (560DF15) for Cy3, and XF1027 (640DF20) for Cy5
    • Single‐band emission filters (Chroma Technologies): S528/38m for Oregon Green; S617/73m for Cy3 and S685/40m for Cy5
  • Additional reagents and equipment for preparing planar bilayers on coverslips ( protocol 3)

Support Protocol 1: Preparation of Cleaned Glass Coverslips for Formation of Planar Bilayers

  Materials
  • Piranha solution (see recipe), freshly prepared
  • 40‐mm glass coverslips (Bioptechs)
  • Polypropylene scissor‐type forceps (Nalge Nunc no. 6320‐0010)

Basic Protocol 3: Immunological Synapse (IS) Formation

  Materials
  • Flow cell containing supported bilayers ( protocol 2)
  • Peptide loading buffer (see recipe)
  • MCC88‐103 peptide sequence, ANERADLIAYLKQATK, or appropriate peptide (Biosynthesis, Inc.; http://www.biosyn.com/)
  • 200 mM phenylmethylsulfonyl fluoride (PMSF) stock in ethanol
  • HBS/HSA buffer (see recipe)
  • 5% casein blocking reagent ( protocol 6)
  • AND TCR‐transgenic T cell blasts, day 7 to 14 (see protocol 7)
  • 20‐ml syringes
  • FCS2 live‐cell micro‐observation system (Bioptechs)
  • Inverted microscope with total internal reflection fluorescence (TIRFM) optics
  • Microscope filters:
    • Dichroic mirror z488‐568‐647rpc (Chroma Technologies): designed to work with the authors' total internal reflection fluorescence microscopy (TIRFM) system with laser lines from Kr‐Ar laser 488, 568, and 647 nm; this element is fixed during experiments and emission filter wheels are changed to detect different fluorescent dyes
    • Single‐band excitation filters from Omega Optical (selected to work with the authors' 150‐W xenon arc lamp): XF1042 (485DF15) for Oregon Green; XF1045 (560DF15) for Cy3, and XF1027 (640DF20) for Cy5
    • Single‐band emission filters (Chroma Technologies): S528/38m for Oregon Green; S617/73m for Cy3 and S685/40m for Cy5.
  • Additional reagents and equipment for inspection of the bilayer (see protocol 2)

Support Protocol 2: Cleaning the Flow Cell

  Materials
  • 70% ethanol
  • Flow cell (Bioptechs; Fig. )

Support Protocol 3: Preparation of 5% Casein Blocking Reagent

  Materials
  • Casein (Sigma cat. no. C5890)
  • 10 N NaOH
  • 10× phosphate‐buffered saline (PBS; see recipe for 1× but prepare at 10× concentration)
  • 1 N NaOH or HCl
  • Ultracentrifuge with 45 Ti rotor (Beckman) or equivalent
  • Ring stand with clamps
  • 35‐ml plastic syringe
  • Thin, stiff polypropylene tubing to fit over an 18‐G to 19‐G needle
  • 18‐G to 19‐G needle
  • 250‐ml, 0.22‐µm Millipore Stericup‐GP filters
  • 5‐ml polystyrene tubes with snap caps

Support Protocol 4: Preparation of T Cell Blasts

  Materials
  • AND TCR transgenic mice on B10 background (The Jackson Laboratory) or TCR transgenic mice of interest
  • B10.Br mice (The Jackson Laboratory)
  • Kanagawa modification of DMEM (see recipe)
  • MCC88‐103 peptide sequence, ANERADLIAYLKQATK, or appropriate peptide (Biosynthesis, Inc.; http://www.biosyn.com/)
  • Recombinant human interleukin‐2 (IL‐2)
  • 75‐cm2 tissue culture flasks
  • Additional reagents and equipment for euthanasia of mice (unit 1.8), harvesting of spleens from mice (unit 1.9), preparation of spleen cell suspensions (unit 3.1), and enrichment of live cells using Lympholyte‐M density gradient (unit 3.22)

Alternate Protocol 1: Use of 6‐Histidine‐Tagged Proteins with Planar Bilayers

  • Liposomes (see protocol 1) prepared from CAP‐biotin‐PE and from ICAM‐1
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Figures

Videos

Literature Cited

Literature Cited
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