Assay for Macrophage‐Mediated Anti‐Tumor Cytotoxicity

George W. Cox1

1 National Cancer Institute, Frederick, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.7
DOI:  10.1002/0471142735.im1407s12
Online Posting Date:  May, 2001
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Abstract

This unit describes a procedure for determining ability of mouse macrophages to lyse tumor cells in vitro. The outlines a three‐stage assay that includes: (1) culture of macrophages (freshly explanted from mice or grown from a cell line) with suspected activating reagents; (2) extensive washing of the cultured macrophages to remove residual reagents, followed by incubation with [111In]‐labeled tumor cells to allow lysis to occur; and (3) collection of cell‐free culture supernatants and measurement of cytolytic activity as a function of 111In released from tumor cells destroyed by activated macrophages. The outlines a method for radiolabeling tumor cells with [111In]oxine.

     
 
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Table of Contents

  • Basic Protocol 1: 111In‐Release Assay for Measuring Macrophage‐Mediated Lysis of Tumor Cells
  • Support Protocol 1: Radiolabeling Tumor Target Cells with [111In]Oxine
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: 111In‐Release Assay for Measuring Macrophage‐Mediated Lysis of Tumor Cells

  Materials
  • Mouse macrophages: from cell line (see Critical Parameters) or freshly explanted (unit 14.1)
  • Hanks balanced salt solution (HBSS; appendix 2A) without Ca2+ or Mg2+, 37°C
  • Complete DMEM‐10or RPMI‐10 medium without 2‐ME ( appendix 2A) made with low‐endotoxin FBS (e.g., HyClone Defined FBS) heat‐inactivated 30 min at 56°C (preheat medium to 37°C just before use)
  • Reagents to be tested for ability to induce anti‐tumor cytotoxicity (unit 14.2)
  • protocol 22.5 × 104 cell/ml [111In]oxine‐labeled tumor cell suspension ( protocol 2)
  • 10% (w/v) SDS or 0.5 M HCl
  • 50‐ml conical polypropylene centrifuge tubes
  • 96‐well U‐bottom microtiter plates (Dynatech) and Linbro plate covers (Flow Laboratories)
  • 8‐channel manifolds, straight and bent (Drummond)
  • Cornwall continuous pipetting outfit (Becton Dickinson Labware)
  • Sorvall RT‐6000 centrifuge with H‐1000B rotor (or equivalent) and microtiter plate carriers
  • Additional reagents and equipment for trypan blue exclusion and Wright‐Giemsa staining ( appendix 3A)
CAUTION:111In is a high‐energy γ radiation–emitting isotope. Use extreme care when handling this isotope; follow standard radiation safety procedures to minimize the extent and duration of exposure to [111In]oxine‐labeled tumor target cells.NOTE: Only a few mouse macrophage cell lines have been shown to exert tumoricidal activity (see Critical Parameters). The tumoricidal mouse macrophage cell lines ANA‐1 and GG2EE are available through the author. Fresh normal macrophages can be obtained from various mouse tissues (unit 14.1).NOTE: Do not use glass or polystyrene cell culture materials when working with macrophages; a distinguishing characteristic of macrophages is their ability to adhere to glass and certain plastics.

Support Protocol 1: Radiolabeling Tumor Target Cells with [111In]Oxine

  Materials
  • Tumor target cells: e.g., P815 cells (ATCC# TIB 64)
  • Complete RPMI‐10 medium ( appendix 2A), 37°C
  • 1 mCi/ml [111In]oxine ([111In]oxyquinoline; Amersham Medi‐Physics)
  • 75‐cm2 tissue culture flasks
  • 50‐ml conical polypropylene centrifuge tubes
  • Sorvall RT‐6000 centrifuge with H‐1000B rotor (or equivalent)
  • Additional reagents and equipment for trypan blue exclusion ( appendix 3A)
CAUTION:111In is a high‐energy γ radiation–emitting isotope. Use extreme care when handling this isotope; follow standard radiation safety procedures to minimize the extent and duration of exposure to [111In]oxine stock solution and [111In]oxine‐labeled target cells.
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Figures

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Literature Cited

Literature Cited
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   Meltzer, M.S. 1981a. Macrophage activation‐quantitation of cytotoxicity by [3H]thymidine release. In Manual of Macrophage Methodology: Collection, Characterization, and Function (H.B. Herscowitz, H.T. Holden, J.A. Bellanti, and A. Ghaffar, eds.) pp. 329‐336. Marcel Dekker, New York.
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   Taramelli, D., Holden, H.T., and Varesio, L. 1980. Endotoxin requirement for macrophage activation by lymphokines in a rapid microcytotoxicity assay. J. Immunol. Methods 37:225‐232.
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   Weinberg, J.B. and J.B. Hibbs, Jr. 1981. Morphological observation of cytotoxicity for tumor cells. In Manual of Macrophage Methodology: Collection, Characterization, and Function (H.B. Herscowitz, H.T. Holden, J.A. Bellanti, and A. Ghaffar, eds.) pp. 345‐355. Marcel Dekker, New York.
   Wiltrout, R.H. and Frost, P. 1980. Cell‐mediated cytotoxic responses induced in vivo and in vitro by a metastatic murine tumor. J. Immunol. 124:2254‐2263.
   Wiltrout, R.H., Frost, P., and Cummings, G.D. 1978. Isotope‐release cytotoxicity assay with the use of indium‐111: Advantage over chromium‐51 in long‐term assays. J. Nat. Cancer Inst. 61:183‐188.
   Wiltrout, R.H., Taramelli, D., and Holden, H.T. 1981a. Measurement of macrophage‐mediated cytotoxicity against adherent and nonadherent target cells by release of 111indium‐oxine. J. Immunol. Methods 43:319‐331.
   Wiltrout, R.H., Taramelli, D., and Holden, H.T. 1981b. Indium‐111 assay of macrophage‐mediated cytolysis. In Manual of Macrophage Methodology: Collection, Characterization, and Function (H.B. Herscowitz, H.T. Holden, J.A. Bellanti, and A. Ghaffar, eds.) pp. 337‐344. Marcel Dekker, New York.
Key References
   Wiltrout et al., 1981a. See above.
  The protocols presented in this unit were reported first in these references.
   Wiltrout et al., 1981b. See above.
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