Measurement of Tumor Cytolysis by Macrophages

Mónica Escórcio‐Correia1, Thorsten Hagemann1

1 Centre for Cancer and Inflammation, Institute of Cancer, Queen Mary University of London, Barts and the London School of Medicine and Dentistry, London, United Kingdom
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.18
DOI:  10.1002/0471142735.im1418s92
Online Posting Date:  February, 2011
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Abstract

This unit describes two different protocols for the measurement of tumor cytolysis by macrophages. Traditionally, cytotoxicity assays have relied on the use of radioactive isotopes. In Basic Protocol 1, cytotoxic activity is measured by the release into the culture supernatant of a radioisotope that had been incorporated by the target cell and is released upon cell death. This poses a problem for some cell lines in which spontaneous isotope release occurs in the absence of effector cell cytotoxicity. In Basic Protocol 2, a nonradioactive approach is used to measure cytolysis that relies on the fluorescence staining of tumor cells with cell‐death markers. It also provides the obvious advantage of avoiding the use of hazardous radioactive materials. Curr. Protoc. Immunol. 92:14.18.1‐14.18.11. © 2011 by John Wiley & Sons, Inc.

Keywords: macrophages; cytolysis; tumor; annexin V; 7‐AAD; CFDA‐SE; [111In]oxine

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of Macrophage‐Mediated Tumor Cell Cytolysis Using [111In]Oxine Radiolabeled Target Cells
  • Basic Protocol 2: Measurement of Macrophage‐Mediated Tumor Cell Cytolysis Using Carboxyfluorescein Diacetate Succinimidyl Ester‐Labeled Target
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Macrophage‐Mediated Tumor Cell Cytolysis Using [111In]Oxine Radiolabeled Target Cells

  Materials
  • Tumor target cells: mouse ovarian cancer cell line ID8 (Roby et al., )
  • RPMI‐10 medium (see recipe)
  • [111In]oxine ([111In]oxyquinoline; Amersham Medi‐Physics)
  • Thioglycollate‐elicited mouse peritoneal macrophages (see unit 14.1)
  • 10% SDS or 0.5 M HCl
  • 15‐ and 50‐ml plastic sterile conical tubes (e.g., BD Falcon)
  • Centrifuge suitable for cell centrifugation
  • 96‐well U‐bottom tissue culture plates with lids
  • γ counter and counting vials
  • Additional reagents and equipment for counting viable cells ( appendix 3B), preparing tumor target cells (unit 7.18), and preparing mouse macrophages (unit 14.1)
CAUTION: [111In]oxine is a radionuclide with high intensity of γ radiation. As with any radioactive isotypes, extreme care should be taken when handling [111In]oxine in order to minimize exposure to harmful radiation.

Basic Protocol 2: Measurement of Macrophage‐Mediated Tumor Cell Cytolysis Using Carboxyfluorescein Diacetate Succinimidyl Ester‐Labeled Target

  Materials
  • Thioglycollate‐elicited mouse peritoneal macrophages (see unit 14.1)
  • Lipopolysaccharide (LPS; Sigma, cat. no. L4391)
  • Interferon γ (IFN‐γ; R&D Systems, cat. no. 485‐MI‐100)
  • Phosphate‐buffered saline (PBS; appendix 2A), prepared using endotoxin‐free reagents
  • Cell dissociation buffer, enzyme‐free, PBS‐based (Invitrogen, cat. no. 13151‐014)
  • RPMI‐10 medium (see recipe)
  • Tumor target cells: nonadherent lymphoma cell line YAC‐1 (ATCC, cat. no. TIB‐160)
  • 5 mM CFDA‐SE stock solution (see recipe)
  • PBS ( appendix 2A) containing 1% (w/v) bovine serum albumin (PBS/5% BSA) or 1% (v/v) fetal bovine serum (PBS/5% FBS)
  • FACS buffer: PBS ( appendix 2A) containing 1% (w/v) bovine serum albumin (BSA)
  • Fc block: purified anti‐mouse CD16/CD32 antibody (BD Bioscience, cat. no. 553142)
  • Allophycocyanin (APC)‐conjugated anti‐mouse F4/80 antibody (F4/80‐APC; eBioscience, cat. no. 17‐4801‐82)
  • Allophycocyanin (APC)‐conjugated anti‐mouse CD45 antibody for single‐color control
  • 10× annexin V binding buffer (BD Biosciences, cat. no. 556454)
  • Annexin V–PE (BD Biosciences, cat. no. 556421)
  • Cell viability solution (ready‐to‐use 7‐AAD solution; BD Biosciences, cat. no. 555816)
  • 140‐mm Petri dishes, sterile but not treated for tissue culture
  • Cell scrapers
  • Centrifuge suitable for cell centrifugation
  • 24‐well tissue culture plates with lids
  • UV cross‐linker (Stratalinker from Stratagene)
  • Additional reagents and equipment for counting viable cells ( appendix 3B) and flow cytometry (Chapter 5)
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Figures

Videos

Literature Cited

Literature Cited
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