In Vivo and Ex Vivo Protocols for Measuring the Killing of Extracellular Pathogens by Macrophages

Eva Medina1, Oliver Goldmann1

1 Infection Immunology Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.19
DOI:  10.1002/0471142735.im1419s92
Online Posting Date:  February, 2011
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Abstract

This unit describes a series of in vivo/ex vivo combined protocols for investigating the interactions (adhesion, phagocytosis, and killing) of extracellular bacteria with peritoneal murine macrophages. It includes steps needed for in vivo infection of murine peritoneal macrophages after intraperitoneal inoculation with the pathogen of interest, as well as the measurement of bacteria associated with or truly internalized by these phagocytic cells. Several protocols for the ex vivo measurement of the ability of peritoneal macrophages to kill the microorganisms that have been ingested during the in vivo infection assay are included. Curr. Protoc. Immunol. 92:14.19.1‐14.19.17. © 2011 by John Wiley & Sons, Inc.

Keywords: macrophages; extracellular pathogens; Streptococcus pyogenes; phagocytosis assay; killing assay; fluorescence microscopy; flow cytometry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of Bacterial Uptake by Peritoneal Macrophages
  • Alternate Protocol 1: Measurement of Macrophages with Associated Bacteria by Fluorescence Microscopy
  • Alternate Protocol 2: Discrimination Between Extracellular and Intracellular Bacteria Using Fluorescence Microscopy
  • Support Protocol 1: Bacterial Inoculum Preparation
  • Support Protocol 2: Preparation of Peritoneal Exudates
  • Basic Protocol 2: Measurement of Bacterial Killing by Peritoneal Macrophages
  • Alternate Protocol 3: Assessment of Bacterial Viability by Fluorescence Microscopy
  • Support Protocol 3: Antibiotic Penetration into Cultured Macrophages
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Bacterial Uptake by Peritoneal Macrophages

  Materials
  • 70% (v/v) ethanol
  • Fluorochrome exhibiting the desired spectral characteristics (e.g., fluorescein isothiocyanate, FITC; Sigma)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Mice
  • Dulbecco's modified Eagle medium, low glucose (DMEM‐LG) containing: glucose (1 g/liter), glutamine (584 mg/liter), sodium pyruvate (110 mg/liter), and 4 mg/liter pyridoxine hydrochloride (store at 4°C)
  • Trypan blue (C 34H 28N 6O 14S 4; diazo dye; Sigma)
  • Antibodies: purified rat anti–mouse CD16/CD32 (Mouse BDFc Block; BD Pharmingen), PE‐conjugated rat anti–mouse F4/80 (eBioscience, cat. no. 12‐4801), and APC‐conjugated rat anti–mouse CD11b (eBioscience, cat. no. 17.0112)
  • 4% paraformaldehyde (see recipe)
  • 1.5‐ml conical polypropylene microcentrifuge tubes (Eppendorf)
  • Microcentrifuge (Eppendorf Centrifuge 5417c)
  • 1‐ml disposable tuberculin syringes fitted with 26 1/2‐G needles (Beckton Dickinson Labware)
  • Dissection board
  • Dissection equipment including:
    • Dissection scissors
    • Tissue forceps
    • Dissecting pins
  • 5‐ml syringe with a 21‐G needle
  • 15‐ml conical polypropylene tubes
  • Refrigerated centrifuge (4°C)
  • Hemacytometer (Neubauer chamber)
  • Glass coverslips
  • Inverse‐phase light microscope
  • FACS tubes (Beckton Dickinson)
  • Vortex
  • Additional reagents and equipment for preparing the bacterial suspension ( protocol 4), euthanizing the animals using CO 2 asphyxiation (unit 1.8), and counting viable cells using trypan blue exclusion ( appendix 3B)

Alternate Protocol 1: Measurement of Macrophages with Associated Bacteria by Fluorescence Microscopy

  • Ammonium chloride, NH 4Cl (Sigma)
  • Mowiol containing 4′,6‐Diamidino‐2‐phenylindol (DAPI; Prolong Gold, Promega)
  • Transparent polish nail
  • 48‐well flat‐bottom tissue culture plates (Nunc)
  • 12‐mm round 0.1‐mm thick glass coverslip
  • Microscope slides (Thermo Scientific)
  • High‐precision tweezers
  • Fluorescence microscope

Alternate Protocol 2: Discrimination Between Extracellular and Intracellular Bacteria Using Fluorescence Microscopy

  • Ammonium chloride, NH 4Cl (Sigma)
  • Albumin from bovine serum, BSA (Sigma)
  • Unconjugated antibodies specific against the pathogen
  • Secondary Alexa Fluor 488 and Alexa Fluor 568 conjugated antibodies (Molecular Probes)
  • Triton X‐100 (Sigma)
  • Mowiol containing DAPI (Prolong Gold, Promega)
  • Transparent polish nail
  • 48‐well flat‐bottom tissue culture plates (Nunc)
  • 12‐mm round 0.1‐mm thick glass coverslip
  • High‐precision tweezers
  • Microscope slides (Thermo Scientific)
  • Paper towels
  • Aluminum foil
  • Fluorescence microscope
NOTE: The concentrations of primary and secondary antibodies must be determined empirically by titration. Excessive primary or secondary antibody will cause “background” staining, while too little will result in a weak signal.

Support Protocol 1: Bacterial Inoculum Preparation

  Materials
  • Stock bacterial culture (e.g., S. pyogenes)
  • THY (Bacto Tryptic Soy Broth supplemented with 0.5 g Bacto yeast extract (Becton Dickinson)
  • Glycerol (Sigma)
  • Blood agar plates (Becton Dickinson)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Spectrophotometer (Novospec II, Pharmacia)

Support Protocol 2: Preparation of Peritoneal Exudates

  • Eliciting agent (e.g., thioglycolate, proteose peptone, and caseine; see reciperecipes and see Table 14.19.1)
    Table 4.9.1   Additional Materials (also see protocol 1)   Additional MaterialsGeneration of Peritoneal Exudates

    Inflammatory stimulant Optimal time for infection Expected yield of total macrophages
    PBS Not applicable 1–2 × 106
    Thioglycolate 4 days 1–2 × 107
    Proteose peptone 3 days 5 × 106
    Caseine 3 days 5 × 106

Basic Protocol 2: Measurement of Bacterial Killing by Peritoneal Macrophages

  Materials
  • Phosphate‐buffered saline (PBS; see recipe)
  • Dulbecco's modified Eagle medium, low glucose (DMEM‐LG) containing: glucose (1 g/liter), glutamine (584 mg/liter), sodium pyruvate (110 mg/liter), and 4 mg/liter pyridoxine hydrochloride (store at 4°C)
  • Gentamicin
  • Triton X‐100
  • 4‐well flat‐bottom tissue culture plates (Nunc)
  • Additional reagents and equipment for preparing infected peritoneal macrophages ( protocol 1)

Alternate Protocol 3: Assessment of Bacterial Viability by Fluorescence Microscopy

  Materials
  • SYTO9 (Invitrogen)
  • Propidium iodide (PI; Invitrogen)
  • Dimethyl sulfoxide (DMSO)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Saponin (Sigma)
  • 3.7% formaldehyde neutral buffer solution (Sigma)
  • Mowiol containing DAPI (Prolong Gold, Promega)
  • Transparent polish nail
  • 24‐well tissue culture plates
  • 12‐mm round 0.1‐mm thick glass coverslip
  • High‐precision tweezers
  • Microscope slides
  • Confocal microscope

Support Protocol 3: Antibiotic Penetration into Cultured Macrophages

  Materials
  • Phosphate‐buffered saline (PBS; see recipe)
  • Dulbecco's modified Eagle medium, low glucose (DMEM‐LG) containing: glucose (1 g/liter), glutamine (584 mg/liter), sodium pyruvate (110 mg/liter), and 4 mg/liter pyridoxine hydrochloride (store at 4°C)
  • Gentamicin
  • 0.1% Triton X‐100
  • Agar plates on which a lawn of bacteria has been previously seeded
  • 4‐well flat‐bottom tissue culture plate
  • 1.5‐ml microcentrifuge tubes
  • Additional reagents and equipment for preparing peritoneal macrophages ( protocol 1)
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Figures

Videos

Literature Cited

Literature Cited
   Drevets, D.A., Canono, B.P., Leenen, P.J., and Campbell, P.A. 1994. Gentamicin kills intracellular Listeria monocytogenes. Infect. Immun. 62:2222‐2228.
   Essmann, F., Bantel, H., Totzke, G., Engels, I.H., Sinha, B., Schulze‐Osthoff, K., and Janicke, R.U. 2003. Staphylococcus aureus alpha‐toxin‐induced cell death: Predominant necrosis despite apoptotic caspase activation. Cell Death Differ. 10:1260‐1272.
   Goldmann, O., Rohde, M., Chhatwal, G.S., and Medina E. 2004. Role of macrophages in host resistance to group A streptococci. Infect. Immun. 72:2956‐2963.
   Goldmann, O., Sastalla, I., Wos‐Oxley, M., Rohde, M., and Medina, E. 2009. Streptococcus pyogenes induces oncosis in macrophages through the activation of an inflammatory programmed cell death pathway. Cell. Microbiol. 11:138‐155.
   Hamrick, T.S., Diaz, A.H., Havell, E.A., Horton, J.R., and Orndorff, P.E. 2003. Influence of extracellular bactericidal agents on bacteria within macrophages. Infect. Immun. 71:1016‐1019.
   Kubica, M., Guzik, K., Koziel, J., Zarebski, M., Richter, W., Gajkowska, B., Golda, A., Maciag‐Gudowska, A., Brix, K., Shaw, L., Foster, T., and Potempa, J. 2008. A potential new pathway for Staphylococcus aureus dissemination: The silent survival of S. aureus phagocytosed by human monocyte‐derived macrophages. PLoS One. 3:e1409.
   Leenen, P.J., de Bruijn, M.F., Voerman, J.S., Campbell, P.A., and van Ewijk, W. 1994. Markers of mouse macrophage development detected by monoclonal antibodies. J. Immunol. Methods 174:5‐19.
   Marriott, H.M., Mitchell, T.J., and Dockrell, D.H. 2008. Pneumolysin: A double‐edged sword during the host‐pathogen interaction. Curr. Mol. Med. 8:497‐509.
   Newman, S.L. and Tucci, M.A. 1990. Regulation of human monocyte/macrophage function by extracellular matrix. Adherence of monocytes to collagen matrices enhances phagocytosis of opsonized bacteria by activation of complement receptors and enhancement of Fc receptor function. J. Clin. Invest. 86:703‐714.
   Pakianathan, D.R. 1995. Extracellular matrix proteins and leukocyte function. J. Leukoc. Biol. 57:699‐702.
   Tabrizi, S.N. and Robins‐Browne, R.M. 1993. Elimination of extracellular bacteria by antibiotics in quantitative assays of bacterial ingestion and killing by phagocytes. J. Immunol. Methods. 158:201‐206.
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