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

  • 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, 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

  • 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, 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)

  • 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

  • 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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Literature Cited

Literature Cited
   Armstrong, J.A. and Hart, P.D. 1971. Response of cultured macrophages to Mycobacterium tuberculosis, with observations on fusion of lysosomes with phagosomes. J. Exp. Med. 134:713‐740.
   Aslan, J E. and Thomas, G. 2009. Death by committee: Organellar trafficking and communication in apoptosis. Traffic 10:1390‐1404.
   Calore, F. , Genisset, C. , Casellato, A. , Rossato, M. , Codolo, G. , Esposti, M.D. , Scorrano, L. , and de Bernard, M. 2010. Endosome‐mitochondria juxtaposition during apoptosis induced by H. pylori VacA. Cell Death Differ. 17:1707‐1716.
   Campbell‐Valois, F.‐X. , Trost, M. , Chemali, M. , Dill, B.D. , Laplante, A. , Duclos, S. , Sadeghi, S. , Rondeau, C. , Morrow, I.C. , Bell, C. , Gagnon, E. , Hatsuzawa, K. , Thibault, P. , and Desjardins, M. 2012. Quantitative proteomics reveals that only a subset of the endoplasmic reticulum contributes to the phagosome. Mol. Cell. Proteom 11:M111.016378.
   Chakraborty, P. , Sturgill‐Koszycki, S. , and Russell, D.G. 1994. Isolation and characterization of pathogen‐containing phagosomes. Methods Cell Biol. 45:261‐276.
   Clemens, D.L. , Lee, B.Y. , and Horwitz, M.A. 2000. Deviant expression of Rab5 on phagosomes containing the intracellular pathogens Mycobacterium tuberculosis and Legionella pneumophila is associated with altered phagosomal fate. Infect. Immun. 68:2671‐2684.
   Coico, R. , McBride, A. , Quarles, J.M. , Stevenson, B. , and Taylor, R.K. 2014. Current Protocols in Microbiology. John Wiley & Sons, Hoboken, N.J.
   Corleis, B. , Korbel, D. , Wilson, R. , Bylund, J. , Chee, R. , and Schaible, U.E. 2012. Escape of Mycobacterium tuberculosis from oxidative killing by neutrophils. Cell. Microbiol. 14:1109‐1121.
   Desjardins, M. , Huber, L.A. , Parton, R.G. , and Griffiths, G. 1994. Biogenesis of phagolysosomes proceeds through a sequential series of interactions with the endocytic apparatus. J. Cell Biol. 124:677‐688.
   Ehrt, S. and Schnappinger, D. 2009. Mycobacterial survival strategies in the phagosome: Defence against host stresses. Cell. Microbiol. 11:1170‐1178.
   Eum, S.‐Y. , Kong, J.‐H. , Hong, M.‐S. , Lee, Y.‐J. , Kim, J.‐H. , Hwang, S.‐H. , Cho, S.‐N. , Via, L.E. , and Barry, C.E. 3rd 2010. Neutrophils are the predominant infected phagocytic cells in the airways of patients with active pulmonary TB. Chest 137:122‐128.
   Garin, J. , Diez, R. , Kieffer, S. , Dermine, J.F. , Duclos, S. , Gagnon, E. , Sadoul, R. , Rondeau, C. , and Desjardins, M. 2001. The phagosome proteome: Insight into phagosome functions. J. Cell Biol. 152:165‐180.
   Gotthardt, D. , Warnatz, H.J. , Henschel, O. , Brückert, F. , Schleicher, M. , and Soldati, T. 2002. High‐resolution dissection of phagosome maturation reveals distinct membrane trafficking phases. Mol. Biol. Cell 13:3508‐3520.
   Grotzke, J.E. , Harriff, M.J. , Siler, A.C. , Nolt, D. , Delepine, J. , Lewinsohn, D.A. , and Lewinsohn, D.M. 2009. The Mycobacterium tuberculosis phagosome is a HLA‐I processing competent organelle. PLoS Pathogens 5:e1000374.
   Herbst, S. , Schaible, U.E. , and Schneider, B.E. 2011. Interferon gamma activated macrophages kill mycobacteria by nitric oxide induced apoptosis. PloS One 6:e19105.
   Hsu, T. , Hingley‐Wilson, S.M. , Chen, B. , Chen, M. , Dai, A.Z. , Morin, P.M. , Marks, C.B. , Padiyar, J. , Goulding, C. , Gingery, M. , Eisenberg, D. , Russell, R.G. , Derrick, S.C. , Collins, F.M. , Morris, S.L. , King, C.H. , and Jacobs, W.R. Jr. 2003. The primary mechanism of attenuation of bacillus Calmette‐Guerin is a loss of secreted lytic function required for invasion of lung interstitial tissue. Proc. Natl. Acad. Sci. U.S.A. 100:12420‐12425.
   Jutras, I. , Houde, M. , Currier, N. , Boulais, J. , Duclos, S. , LaBoissière, S. , Bonneil, E. , Kearney, P. , Thibault, P. , Paramithiotis, E. , Hugo, P. , and Desjardins, M. 2008. Modulation of the phagosome proteome by interferon‐gamma. Mol. Cell. Proteom. 7:697‐715.
   Kelley, V.A. and Schorey, J.S. 2003. Mycobacterium's arrest of phagosome maturation in macrophages requires Rab5 activity and accessibility to iron. Mol. Biol. Cell 14:3366‐3377.
   Kelley, V.A. and Schorey, J.S. 2004. Modulation of cellular phosphatidylinositol 3‐phosphate levels in primary macrophages affects heat‐killed but not viable Mycobacterium avium's transport through the phagosome maturation process. Cell. Microbiol. 6:973‐985.
   Klug, K. , Ehlers, S. , Uhlig, S. , and Reiling, N. 2011. Mitogen‐activated protein kinases p38 and ERK1/2 regulated control of Mycobacterium avium replication in primary murine macrophages is independent of tumor necrosis factor‐α and interleukin‐10. Innate Immun. 17:470‐485.
   Korbel, D.S. , Schneider, B.E. , and Schaible, U.E. 2008. Innate immunity in tuberculosis: Myths and truth. Microbes Infect. 10:995‐1004.
   Kumar, D. and Rao, K.V.S. 2011. Regulation between survival, persistence, and elimination of intracellular mycobacteria: A nested equilibrium of delicate balances. Microbes Infect. 13:121‐133.
   Lee, B.‐Y. , Jethwaney, D. , Schilling, B. , Clemens, D.L. , Gibson, B.W. , and Horwitz, M.A. 2010. The Mycobacterium bovis bacille Calmette‐Guerin phagosome proteome. Mol. Cell. Proteomics 9:32‐53.
   Martin, C.J. , Booty, M.G. , Rosebrock, T.R. , Nunes‐Alves, C. , Desjardins, D.M. , Keren, I. , Fortune, S.M. , Remold, H.G. , and Behar, S.M. 2012. Efferocytosis is an innate antibacterial mechanism. Cell Host Microbe 12:289‐300.
   Matsumoto, A. 1981. Isolation and electron microscopic observations of intracytoplasmic inclusions containing Chlamydia psittaci . J. Bacteriol. 145:605‐612.
   Matsumoto, A. , Bessho, H. , Uehira, K. , and Suda, T. 1991. Morphological studies of the association of mitochondria with chlamydial inclusions and the fusion of chlamydial inclusions. J. Electron Microsc. 40:356‐363.
   Podinovskaia, M. , Lee, W. , Caldwell, S. , and Russell, D.G. 2013. Infection of macrophages with Mycobacterium tuberculosis induces global modifications to phagosomal function. Cell. Microbiol. 15:843‐859.
   Pokkali, S. and Das, S.D. 2009. Augmented chemokine levels and chemokine receptor expression on immune cells during pulmonary tuberculosis. Hum. Immunol. 70:110‐115.
   Reiling, N. , Blumenthal, A. , Flad, H.D. , Ernst, M. , and Ehlers, S. 2001. Mycobacteria‐induced TNF‐alpha and IL‐10 formation by human macrophages is differentially regulated at the level of mitogen‐activated protein kinase activity. J. Immunol. 167:3339‐3345.
   Reiling, N. , Homolka, S. , Walter, K. , Brandenburg, J. , Niwinski, L. , Ernst, M. , Herzmann, C. , Lange, C. , Diel, R. , Ehlers, S. , and Niemann, S. 2013. Clade‐specific virulence patterns of Mycobacterium tuberculosis complex strains in human primary macrophages and aerogenically infected mice. MBio 4. Jul 30;4(4). pii: e00250‐13. doi: 10.1128/mBio.00250‐13.
   Roberts, E.A. , Chua, J. , Kyei, G.B. , and Deretic, V. 2006. Higher order Rab programming in phagolysosome biogenesis. J. Cell Biol. 174:923‐929.
   Russell, D.G. , Vanderven, B.C. , Glennie, S. , Mwandumba, H. , and Heyderman, R.S. 2009. The macrophage marches on its phagosome: Dynamic assays of phagosome function. Nat. Rev. Immunol. 9:594‐600.
   Sanjuan, M.A. , Dillon, C.P. , Tait, S.W.G. , Moshiach, S. , Dorsey, F. , Connell, S. , Komatsu, M. , Tanaka, K. , Cleveland, J.L. , Withoff, S. , and Green, D.R. 2007. Toll‐like receptor signalling in macrophages links the autophagy pathway to phagocytosis. Nature 450:1253‐1257.
   Schaible, U.E. , Winau, F. , Sieling, P.A. , Fischer, K. , Collins, H.L. , Hagens, K. , Modlin, R.L. , Brinkmann, V. , and Kaufmann, S.H.E. 2003. Apoptosis facilitates antigen presentation to T lymphocytes through MHC‐I and CD1 in tuberculosis. Nat. Med, 9:1039‐1046.
   Schneider‐Brachert, W. , Tchikov, V. , Neumeyer, J. , Jakob, M. , Winoto‐Morbach, S. , Held‐Feindt, J. , Heinrich, M. , Merkel, O. , Ehrenschwender, M. , Adam, D. , Mentlein, R. , Kabelitz, D. , and Schütze, S. 2004. Compartmentalization of TNF receptor 1 signaling: Internalized TNF receptosomes as death signaling vesicles. Immunity 21:415‐428.
   Schneider‐Brachert, W. , Tchikov, V. , Merkel, O. , Jakob, M. , Hallas, C. , Kruse, M.‐L. , Groitl, P. , Lehn, A. , Hildt, E. , Held‐Feindt, J. , Dobner, T. , Kabelitz, D. , Krönke, M. , and Schütze, S. 2006. Inhibition of TNF receptor 1 internalization by adenovirus 14.7K as a novel immune escape mechanism. J. Clin. Invest. 116:2901‐2913.
   Schütze, S. , Tchikov, V. , Kabelitz, D. , and Kroenke, M. 2003. Vorrichtung zur Isolierung von an magnetischen Partikeln gebundenem biologischen Material in einem Magnetfeld. German patent no. DE 10144291 C2.
   Schütze, S. , Tchikov, V. , and Schneider‐Brachert, W. 2008. Regulation of TNFR1 and CD95 signalling by receptor compartmentalization. Nat. Rev. Mol. Cell Biol. 9:655‐662.
   Seto, S. , Tsujimura, K. , and Koide, Y. 2011. Rab GTPases regulating phagosome maturation are differentially recruited to mycobacterial phagosomes. Traffic 12:407‐420.
   Soldati, T. and Neyrolles, O. 2012. Mycobacteria and the intraphagosomal environment: take it with a pinch of salt(s)! Traffic 13:1042‐1052.
   Steinhäuser, C. , Heigl, U. , Tchikov, V. , Schwarz, J. , Gutsmann, T. , Seeger, K. , Brandenburg, J. , Fritsch, J. , Schroeder, J. , Wiesmüller, K.‐H. , Rosenkrands, I. , Walther, P. , Pott, J. , Krause, E. , Ehlers, S. , Schneider‐Brachert, W. , Schütze, S. , and Reiling, N. 2013. Lipid‐labeling facilitates a novel magnetic isolation procedure to characterize pathogen‐containing phagosomes. Traffic 14:321‐336.
   Stuart, L.M. , Boulais, J. , Charriere, G.M. , Hennessy, E.J. , Brunet, S. , Jutras, I. , Goyette, G. , Rondeau, C. , Letarte, S. , Huang, H. , Ye, P. , Morales, F. , Kocks, C. , Bader, J.S. , Desjardins, M. , and Ezekowitz, R.A. 2007. A systems biology analysis of the Drosophila phagosome. Nature 445:95‐101.
   Tchikov, V. and Schütze, S. 2008. Immunomagnetic isolation of tumor necrosis factor receptosomes. Methods Enzymol. 442:101‐123.
   Tchikov, V. , Fritsch, J. , Kabelitz, D. , and Schütze, S. 2010. Immunomagnetic isolation of subcellular compartments. In Immunology of Infection ( S.H.E. Kaufmann and Dieter Kabelitz , eds.) pp. 21‐33. Academic Press, Waltham, Mass.
   Tchikov, V. , Fritsch, J. , and Schütze, S. 2013. Separation of magnetically isolated TNF‐receptosomes from mitochondria. Methods Enzymol. 535:327‐349.
   Touret, N. , Paroutis, P. , Terebiznik, M. , Harrison, R.E. , Trombetta, S. , Pypaert, M. , Chow, A. , Jiang, A. , Shaw, J. , Yip, C. , Moore, H.P. , van der Wel, N. , Houben, D. , Peters, P.J. , de Chastellier, C. , Mellman, I. , and Grinstein, S. 2005. Quantitative and dynamic assessment of the contribution of the ER to phagosome formation. Cell 123:157‐170.
   Trost, M. , English, L. , Lemieux, S. , Courcelles, M. , Desjardins, M. , and Thibault, P. 2009. The phagosomal proteome in interferon‐gamma‐activated macrophages. Immunity 30:143‐154.
   Tsai, M.C. , Chakravarty, S. , Zhu, G. , Xu, J. , Tanaka, K. , Koch, C. , Tufariello, J. , Flynn, J. , and Chan, J. 2006. Characterization of the tuberculous granuloma in murine and human lungs: cellular composition and relative tissue oxygen tension. Cell. Microbiol. 8:218‐232.
   Van der Wel, N. , Hava, D. , Houben, D. , Fluitsma, D. , van Zon, M. , Pierson, J. , Brenner, M. , and Peters, P.J. 2007. M. tuberculosis and M. leprae translocate from the phagolysosome to the cytosol in myeloid cells. Cell 129:1287‐1298.
   Via, L.E. , Deretic, D. , Ulmer, R.J. , Hibler, N.S. , Huber, L.A. , and Deretic, V. 1997. Arrest of mycobacterial phagosome maturation is caused by a block in vesicle fusion between stages controlled by rab5 and rab7. J. Biol. Chem. 272:13326‐13331.
   Welin, A. and Lerm, M. 2012. Inside or outside the phagosome? The controversy of the intracellular localization of Mycobacterium tuberculosis . Tuberculosis 92:113‐120.
   West, A.P. , Brodsky, I.E. , Rahner, C. , Woo, D.K. , Erdjument‐Bromage, H. , Tempst, P. , Walsh, M.C. , Choi, Y. , Shadel, G.S. , and Ghosh, S. 2011a. TLR signalling augments macrophage bactericidal activity through mitochondrial ROS. Nature 472:476‐480.
   West, A.P. , Shadel, G.S. , and Ghosh, S. 2011b. Mitochondria in innate immune responses. Nat. Rev. Immunol. 11:389‐402.
   WHO. 2012. Global Tuberculosis Report. World Health Organization (WHO). Available at: [Accessed November 26, 2012].
   Winau, F. , Weber, S. , Sad, S. , de Diego, J. , Hoops, S.L. , Breiden, B. , Sandhoff, K. , Brinkmann, V. , Kaufmann, S.H.E. , and Schaible, U.E. 2006. Apoptotic vesicles crossprime CD8 T cells and protect against tuberculosis. Immunity 24:105‐117.
   Wirth, T. , Hildebrand, F. , Allix‐Béguec, C. , Wölbeling, F. , Kubica, T. , Kremer, K. , van Soolingen, D. , Rüsch‐Gerdes, S. , Locht, C. , and Brisse, S. 2008. Origin, spread and demography of the Mycobacterium tuberculosis complex. PLoS Pathogens 4:e1000160.
   Xu, Y. , Loison, F. , and Luo, H.R. 2010. Neutrophil spontaneous death is mediated by down‐regulation of autocrine signaling through GPCR, PI3Kgamma, ROS, and actin. Proc. Natl. Acad. Sci. U.S.A. 107:2950‐2955.
   Yazdanpanah, B. , Wiegmann, K. , Tchikov, V. , Krut, O. , Pongratz, C. , Schramm, M. , Kleinridders, A. , Wunderlich, T. , Kashkar, H. , and Utermöhlen, O. 2009. Riboflavin kinase couples TNF receptor 1 to NADPH oxidase. Nature 460:1159‐1163.
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