Measuring TLR Function in Transfectants

Prasad Rallabhandi1

1 U.S. Food and Drug Administration, Laurel, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.16
DOI:  10.1002/0471142735.im1416s91
Online Posting Date:  November, 2010
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Abstract

This unit summarizes a combination of methods that can be optimized for measuring toll‐like receptor (TLR) function. TLRs serve as primary innate immune sensors and exhibit high specificity towards evolutionarily conserved microbial and viral structures. The unit focuses specifically on TLR4, the principal Gram‐negative lipopolysaccharide (LPS) sensor. Methods described include transient transfections, analyses of activation of various promoters in reporter‐gene assays, and induction of IL‐8 secretion. Other topics that will be briefly discussed include the necessity for the assessment of surface expression of transmembrane receptors (e.g., TLR4) using FACS analysis, and a permutation of the TLR functional analysis approach using site‐directed mutagenesis. Curr. Protoc. Immunol. 91:14.16.1‐14.16.15. © 2010 by John Wiley & Sons, Inc.

Keywords: toll‐like receptors (TLRs); TLR agonists; lipopolysaccharide (LPS); signal transduction; cell activation; transient transfection; reporter or luciferase assays; mutants; FACS analysis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of TLR4‐Expressing HEK293T Cells Using Transient Transfection
  • Support Protocol 1: Luciferase and β‐Galactosidase Reporter Gene Assays
  • Support Protocol 2: Transformation of E. coli and Endotoxin‐Free Plasmid Preparation
  • Alternate Protocol 1: Quantification of Agonist‐Induced IL‐8 Secretion as a Measure of TLR Function in Transfectants
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of TLR4‐Expressing HEK293T Cells Using Transient Transfection

  Materials
  • HEK293T cells (ATCC no. CRL‐11268)
  • 1× complete DMEM (see recipe)
  • 0.25% trypsin/1 mM EDTA (Invitrogen)
  • Serum‐free DMEM (Invitrogen)
  • Plasmids and human version gene expression constructs ( protocol 3): pcDNA3‐TLR4 (untagged‐TLR4, wild‐type), pFLAG‐CMV1‐TLR4 (N‐terminal FLAG‐tagged TLR4, wild‐type), pcDNA3‐CD14, pEFBOS‐His‐MD‐2, pEFBOS‐HA‐MD‐2, and pELAM‐luciferase (pELAM‐luc), can be obtained from Addgene, and the expression vector pCMV1‐β‐galactosidase can be obtained from Clontech; D299G, T399I, and D299G/T399I mutations were introduced into the extracellular domain of both untagged‐ and FLAG‐tagged‐TLR4 by site‐directed mutagenesis, using a Quick‐Change Site‐Directed Mutagenesis kit (Stratagene), as described briefly in Background Information
  • pcDNA3 empty vector (Invitrogen)
  • SuperFect transfection reagent (Qiagen)
  • Phosphate‐buffered saline (PBS; appendix 2A), cold
  • Lipopolysaccharide (LPS, protein‐free, phenol/water‐extracted, free of TLR2‐activating contaminants (InvivoGen, http://www.invivogen.com/)
  • Reporter Lysis 5× Buffer (Promega; part of Luciferase Assay System)
  • 75‐ and 150‐cm2 tissue culture flasks (Corning)
  • Tabletop centrifuge (e.g., Beckman CS‐6R centrifuge)
  • 12‐well culture plates (BD Biosciences)
  • 14‐ml round‐bottom tubes (BD Falcon, cat. no. 352006)
  • 0.5‐ml and 1‐ml microcentrifuge tubes
  • Additional reagents and equipment for counting viable cells by trypan blue exclusion ( appendix 3B) and measuring luciferase and β‐galactosidase activity ( protocol 2)

Support Protocol 1: Luciferase and β‐Galactosidase Reporter Gene Assays

  Materials
  • Luciferase Assay System (Promega)
  • TLR4‐transfected HEK293T cell lysates ( protocol 1)
  • Galacto‐Light System (Applied Biosystems)
  • Light Emission Accelerator (Applied Biosystems)
  • BioRad Protein Assay Dye Reagent Concentrate
  • Luminometer and luminometer tubes (e.g., Berthold LB 9507; Berthold Technologies, http://www.berthold.com)
  • Statistical software package (e.g., GraphPad Prism; http://www.graphpad.com)

Support Protocol 2: Transformation of E. coli and Endotoxin‐Free Plasmid Preparation

  Materials
  • Sub‐cloning efficiency DH5‐α cells (Invitrogen)
  • Plasmid DNA of interest (see unit introduction and protocol 1 materials list)
  • SOC ( appendix 3N) or LB medium (unit 10.18)
  • LB agar/ampicillin plates (or other selective LB plates as required by the plasmid)
  • EndoFree Plasmid Maxi Kits (Qiagen)
  • 42°C water bath
  • 37°C shaking incubator

Alternate Protocol 1: Quantification of Agonist‐Induced IL‐8 Secretion as a Measure of TLR Function in Transfectants

  Materials
  • HEK293T cells (ATCC no. CRL‐11268)
  • 1× complete DMEM (see recipe)
  • Serum‐free DMEM (Invitrogen)
  • Plasmids and DNA constructs ( protocol 3): pcDNA3‐hTLR4, FLAG‐CMV1‐TLR4, pcDNA3‐CD14, and pEFBos‐HA‐MD2 (Addgene), or other gene constructs
  • pCDNA3 empty vector (Invitrogen)
  • SuperFect transfection reagent (Qiagen)
  • Phosphate‐buffered saline (PBS; appendix 2A), cold
  • Lipopolysaccharide (LPS, protein‐free, phenol/water‐extracted, free of TLR2‐activating contaminants (InvivoGen, http://www.invivogen.com/)
  • 24‐well tissue culture plates (BD Biosciences)
  • 14‐ml round‐bottom tubes (BD Falcon, cat. no. 352006)
  • Additional reagents and equipment for ELISA (unit 2.1)
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Figures

Videos

Literature Cited

Literature Cited
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