Immunomagnetic Isolation of Pathogen‐Containing Phagosomes and Apoptotic Blebs from Primary Phagocytes

Christine Steinhäuser1, Tobias Dallenga2, Vladimir Tchikov3, Ulrich E. Schaible2, Stefan Schütze3, Norbert Reiling1

1 Division of Microbial Interface Biology, Research Center Borstel, Leibniz Center for Medicine and Biosciences, Borstel, 2 Division of Cellular Microbiology, Research Center Borstel, Leibniz Center for Medicine and Biosciences, Borstel, 3 Institute of Immunology, Christian‐Albrechts‐University of Kiel, Kiel
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.36
DOI:  10.1002/0471142735.im1436s105
Online Posting Date:  April, 2014
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Abstract

Macrophages and polymorphonuclear neutrophils are professional phagocytes essential in the initial host response against intracellular pathogens such as Mycobacterium tuberculosis. Phagocytosis is the first step in phagocyte‐pathogen interaction, where the pathogen is engulfed into a membrane‐enclosed compartment termed a phagosome. Subsequent effector functions of phagocytes result in killing and degradation of the pathogen by promoting phagosome maturation, and, terminally, phago‐lysosome fusion. Intracellular pathogenic microbes use various strategies to avoid detection and elimination by phagocytes, including induction of apoptosis to escape host cells, thereby generating apoptotic blebs as shuttles to other cells for pathogens and antigens thereof. Hence, phagosomes represent compartments where host and pathogen become quite intimate, and apoptotic blebs are carrier bags of the pathogen's legacy. In order to investigate the molecular mechanisms underlying these interactions, both phagosomes and apoptotic blebs are required as purified subcellular fractions for subsequent analysis of their biochemical properties. Here, we describe a lipid‐based procedure to magnetically label surfaces of either pathogenic mycobacteria or apoptotic blebs for purification by a strong magnetic field in a novel free‐flow system. Curr. Protoc. Immunol. 105:14.36.1‐14.36.26. © 2014 by John Wiley & Sons, Inc.

Keywords: macrophages; primary cells; mycobacteria; phagosomes; apoptotic vesicles; isolation protocol

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation and Characterization of Mycobacteria‐Containing Phagosomes Using a Free‐Flow Magnetic Chamber System
  • Basic Protocol 2: Isolation and Characterization of Apoptotic Blebs
  • Support Protocol 1: Cultivation of Mycobacteria, Generation of Frozen Aliquots, and Determination of Colony‐Forming Units (CFU)
  • Support Protocol 2: Generation of Apoptotic PMNS and Macrophages from Human Peripheral Blood
  • Reagents and Solutions
  • Commentary
  • Acknowledgments
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation and Characterization of Mycobacteria‐Containing Phagosomes Using a Free‐Flow Magnetic Chamber System

  Materials
  • Frozen aliquots of M. avium (strain SE01) or M. tuberculosis (H37Rv, ATCC no. 27294 ( protocol 3; also see Reiling et al., , )
  • Phosphate‐buffered saline (PBS; PAA, Pasching, Austria, cat. no. H15‐011)
  • Lipobiotin (LB, PHCKKKKK(Aca‐Aca‐Biotin) x 3 TFA, N‐Palmitoyl‐S‐(1,2‐bishexadecyloxy‐carbonyl) ethyl‐[R]‐cysteinyl‐[S]‐lysyl‐[S]‐lysyl‐[S]‐lysyl‐[S]‐lysyl ‐[S]‐lysine(ϵ‐aminocaproyl‐ϵ‐aminocaproyl‐biotinyl) x 3 CF3COOH (EMC Microcollections GmbH): add 1 mg lyophilized powder to 1 ml water for injection (B. Braun) and disperse in an ultrasonic bath (store up to 3 months at 4°C; do not freeze)
  • Streptavidin‐Cy5 (Dianova, cat. no. 016‐170‐084)
  • Flow cytometry buffer: phosphate‐buffered saline (PBS; PAA, Pasching, Austria, cat. no. H15‐011) containing 3% FBS and 0.1% (w/v) NaN 3
  • Streptavidin‐conjugated magnetic nanoparticles (MagCellect, R&D Systems, size, 150 nm, cat. no. MAG999)
  • Murine bone‐marrow derived macrophages (see unit )
  • Murine macrophage medium (see recipe), with and without 50 M‐CSF
  • M‐CSF (R&D systems, cat. no. 216‐MC‐005; Klug et al., )
  • Alfazyme solution (PAA, Pasching, Austria; cat. no. L11‐012)
  • Serum‐free DMEM medium (unsupplemented)
  • Homogenization buffers (see recipes): HB1, HB2, HB3, and HB4
  • Disodium EDTA (Carl Roth, cat. no. 8043.2)
  • Benzonase Endonuclease (Novagen, cat. no. 70664‐3)
  • Cytochalasin D (Calbiochem, cat. no. 250255‐1MG)
  • 1:1 ethanol/H 2O mixture supplemented with 0.1% (v/v) dishwashing liquid
  • Bench top centrifuge (e.g., Hettich Rotanta with a swing‐out rotor for the use with 50‐ml and 1‐ml tubes, and a plate adaptor)
  • Test tube shaker (e.g., Heidolph REAX, cat.no 541‐10000‐00)
  • 12‐ml flow cytometry tubes (BD Falcon Cat. no. 352054)
  • Flow cytometer (e.g., FACS Canto II, BD Biosciences; also see Chapter 5)
  • Petri dishes (10‐cm diameter; Sarstedt, cat. no. 82.1472 or Nunc, cat. no. 150350)
  • 50‐ml tubes for cell culture (Corning, cat. no. 430829)
  • 1.5‐ml screw‐cap micro test tubes (Sarstedt, Cat. no. 72.690.001)
  • Sonication device: closed cylinder (Cup Horn “High Intensity,” G. Heinemann, cat. no. 101‐147‐046) working as a cup resonator connected to a transducer (Branson Sonifier 450 II Classic; G. Heinemann, cat. no. 101‐063‐675)
  • Free‐flow magnetic chamber HOKImag (HOOCK GmbH, Kiel, Germany)
  • Peristaltic pump (Minipulse 3; Gilson) with speed control module R2 medium flow pump head (Gilson, cat. no. F117800)
  • Tubing for pump head (ISMAPRENE tube with 2 stoppers; Phar Med, cat. no. SC0323)
  • Flow column (HOOCK GmbH)
  • Collecting device (HOOCK GmbH)
  • 15‐ml tubes for cell culture (Corning, cat. no. 430791)
  • Teflon or ceramic scissors
  • Plunger of 1‐ml syringe
  • Additional reagents and equipment for flow cytometry (Chapter 5) and staining for viable cells by trypan blue exclusion ( )

Basic Protocol 2: Isolation and Characterization of Apoptotic Blebs

  Materials
  • Apoptotic PMN culture ( protocol 4)
  • Phosphate‐buffered saline (PBS; PAA, Pasching, Austria, cat. no. H15‐011)
  • Fetal bovine serum (FBS; Bichrom. cat. no. S0115)
  • DNase I (Sigma‐Aldrich, cat. no. D5025)
  • Lipobiotin (LB, PHCKKKKK(Aca‐Aca‐Biotin) x 3 TFA, N‐Palmitoyl‐S‐(1,2‐bishexadecyloxy‐carbonyl) ethyl‐[R]‐cysteinyl‐[S]‐lysyl‐[S]‐lysyl‐[S]‐lysyl‐[S]‐lysyl ‐[S]‐lysine(ϵ‐aminocaproyl‐ϵ‐aminocaproyl‐biotinyl) x 3 CF3COOH (EMC Microcollections GmbH): add 1 mg lyophilized powder to 1 ml water for injection (B. Braun) and disperse in an ultrasonic bath (store up to 3 months at 4°C; do not freeze)
  • Carboxyfluorescein diacetate, succinimidyl ester (CFDA SE; Invitrogen, Vybrant, cat. no. V12883; optional)
  • Complete medium for human PMN/macrophage culture (see recipe)
  • Streptavidin‐Cy5 (Dianova, cat. no. 016‐170‐084)
  • Human macrophages seeded on glass coverslips in wells of 24‐well plate ( protocol 4)
  • 4% (w/v) paraformaldehyde (PFA, Carl Roth, cat. no. 0335.3)
  • Permeabilization buffer: 0.05% (v/v) Triton‐X 100 (Carl Roth, cat. no. 3051.3) in PBS (PAA, Pasching, Austria, cat. no. H15‐011)
  • Blocking buffer: 10% normal goat serum (PAA, Pasching, Austria, cat. no. B11‐035) in PBS (PAA, Pasching, Austria, cat. no. H15‐011)
  • Streptavidin‐Cy3 (Invitrogen, cat. no. 434315)
  • Confocal‐Matrix mounting medium (Micro‐Tech‐Lab)
  • Streptavidin‐conjugated magnetic nanoparticles (MagCellect, R&D Systems, size, 150 nm, cat. no. MAG999)
  • 50‐ml tubes for cell culture (Corning, cat.no. 430829)
  • Centrifuge with swing‐out rotor accommodating 50‐ and 15‐ml tubes
  • Ultracentrifuge tubes, capacity 10.4 ml (Beckman Coulter, cat. no. 355603)
  • Ultracentrifuge (Beckman L7‐55 and Type 70.1 Ti rotor)
  • 1.5‐ml micro test tube (Sarstedt, Cat. no. 72.692.005)
  • Round‐bottom tube suitable for flow cytometer (BD Falcon)
  • Flow cytometer (e.g. LSR II, BD Bioscience)
  • Glass microscope slides
  • Confocal or fluorescence microscope
  • Additional reagents and equipment for preparation of apoptotic human PMN culture and human macrophages ( protocol 4) and magnetic isolation ( protocol 1)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper aseptic technique should be used accordingly.NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: Cultivation of Mycobacteria, Generation of Frozen Aliquots, and Determination of Colony‐Forming Units (CFU)

  Materials
  • Mycobacterial strain of interest (e.g., M. tuberculosis H37Rv, M. avium SE01, or M. bovis BCG)
  • Middlebrook 7H9 liquid medium (see recipe)
  • Phosphate‐buffered saline (PBS; PAA, Pasching, Austria, cat. no. H15‐011)
  • 0.05% (v/v) Tween 80, sterilized by autoclaving
  • Middlebrook 7H10 agar plates (see recipe) supplemented with 10% (v/v) bovine serum (BioWest, France)
  • Sterile square media bottles (VWR, cat. no 215‐6700H)
  • Spectrophotometer
  • Roller‐bottle incubation system (Wheaton)
  • Multi‐mode microplate reader (e.g. Biotek, Synergy II)
  • Refrigerated centrifuge
  • 1‐ml syringe with 26‐G needle
  • Additional reagents and equipment for spectrophotometric monitoring of bacterial growth (unit ) and growth and manipulation of mycobacteria (see Chapter 10 in Coico et al., )

Support Protocol 2: Generation of Apoptotic PMNS and Macrophages from Human Peripheral Blood

  Materials
  • Human peripheral blood, freshly drawn
  • Histopaque 1119 (Sigma‐Aldrich, cat. no. 11191‐6×100ML)
  • Phosphate‐buffered saline (PBS; PAA, Pasching, Austria, cat. no. H15‐011)
  • Percoll (Sigma‐Aldrich, cat. no. P1644‐500ML)
  • MACS buffer (see recipe)
  • Anti‐human CD14+ magnetic beads (Miltenyi Biotec, cat. no. 130‐050‐201)
  • M‐CSF (R&D systems, cat. no. 216‐MC‐005)
  • 15‐ and 50‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Refrigerated centrifuge
  • Neubauer counting chamber (see )
  • LS column (Miltenyi Biotec, cat. no. 130.042.401)
  • MACS magnet (Mini MACS, Miltenyi Biotec)
  • Glass coverslips (sterile)
  • 24‐well culture plates
  • Additional reagents and equipment for counting cells ( ) and counting viable cells by trypan blue exclusion ( )
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Figures

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Literature Cited

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