Measurement of Tumor Necrosis Factor and Lymphotoxins

M. Michele Hogan1, Stefanie N. Vogel2

1 The American Association of Immunologists, Rockville, Maryland, 2 University of Maryland School of Medicine, Baltimore, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.10
DOI:  10.1002/cpim.23
Online Posting Date:  April, 2017
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Abstract

The tumor necrosis factor (TNF) superfamily of cytokines plays critical roles in all aspects of the immune response. TNF and the lymphotoxins (LT), LTα and LTβ, are particularly important as major effector cytokines and mediators or lymphoid organ development. One of the classical methods for the measurement of TNF and LTα activity is by demonstrating their ability to lyse certain target cells. A detailed protocol for the measurement of this activity using a highly sensitive indicator cell line is presented. More recently, ELISA assays have been developed to measure the protein concentration of these cytokines in any type of biologic fluid. © 2017 by John Wiley & Sons, Inc.

Keywords: TNF; lymphotoxin; cell death

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Cell Death Assay for the Detection of TNF/LT‐α
  • Alternate Protocol 1: Measurement of TNF and Lymphotoxins by ELISA Assays
  • Support Protocol 1: Propagation and Preparation of L929 Fibroblasts
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Cell Death Assay for the Detection of TNF/LT‐α

  Materials
  • TNF‐sensitive L929 fibroblasts ( protocol 3Support Protocol)
  • Supplemented Eagles minimum essential medium (EMEM; unit ; Vogel et al., )
  • Actinomycin D solution
  • 0.9% (w/v) saline (Abbott)
  • 0.05% crystal violet (w/v) in 20% ethanol
  • 100% methanol
  • 96‐well flat‐bottom microtiter plate (Falcon, cat. no. 3072 or Costar, cat. no. 3596)
  • 8‐well aspirator (Drummond; e.g., Thomas Scientific cat. no. 7691‐R32)
  • 8‐channel pipettor (fixed or adjustable to 50 µl and 100 µl; e.g., Costar Octapette #4850, #4800)
  • Microtiter plate reader with 595‐nm filter (optional)

Alternate Protocol 1: Measurement of TNF and Lymphotoxins by ELISA Assays

  Additional Materials (also see protocol 1Basic Protocol)
  • L929 fibroblasts (see critical parameters)
  • Trypsin solution (unit ; Vogel et al., )
  • 150‐cm2 tissue culture flask
  • 50‐ml conical centrifuge tube
  • Additional reagents and equipment for cell counting and evaluating cell viability by trypan blue exclusion ( ; Strober, )
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Figures

Videos

Literature Cited

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