We thank members of the Phagosome Biology Laboratory for critical reading of the manuscript. This work is supported by a Helmholtz Young Investigator grant (Initiative and Networking funds of the Helmholtz Association) and a Priority Program SPP1580 Grant of the German Research Council (Deutsche Forschungsgemeinschaft, DFG).

Animal care and all procedures in this protocol follow the institutional and national guidelines.
Prepare the preparations that are indicated by &#34;&#928;-&#34; beforehand.
1. Preparation of BMMs
<ol>
	<li>Perform the culling of the mice by cervical dislocation.</li>
	<li>Remove femur and tibia bones, free bones from tissue and trim both ends for the accessibility of the bone marrow.</li>
	<li>Keep bones in ice-cold PBS or ice-cold DMEM medium (supplemented with...

<ol>
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Biol. 21, 511-527 (2005).</li><li>Iwasaki, A., Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+adaptive+immunity+by+the+innate+immune+system.">Regulation of adaptive immunity by the innate immune system.</a> Science. 327, 291-295 (2010).</li><li>Stuart, L., Ezekowitz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis:+elegant+complexity.">Phagocytosis: elegant complexity.</a> Immunity. 22, 539-550 (2005).</li><li>Blander, J. M., Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+regulation+of+phagosome+maturation+and+antigen+presentation.">On regulation of phagosome maturation and antigen presentation.</a> Nat. Immunol. 7, 1029-1035 (2006).</li><li>Flannagan, R. 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Immunol. 14, 103-110 (2002).</li><li>Bohdanowicz, M., Cos&#237;o, G., Backer, J., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Class+I+and+class+III+phosphoinositide+3-kinases+are+required+for+actin+polymerization+that+propels+phagosomes.">Class I and class III phosphoinositide 3-kinases are required for actin polymerization that propels phagosomes.</a> J. Cell Biol. 191, 999-1012 (2010).</li><li>Hoffmann, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+receptor-ligand+interactions+modulate+gene+expression+and+phagosomal+properties+during+both+early+and+late+stages+of+phagocytosis.">Initial receptor-ligand interactions modulate gene expression and phagosomal properties during both early and late stages of phagocytosis.</a> Eur. J. 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In the following section we will discuss critical steps of the presented method and its limitations. Further, we will connect some of the common problems with their solutions, introduce possible modifications and consider advantages of our method as well as suitable complementary methods for this approach.
The preparation of the beads, including the coupling of IgG to the bead surface, is crucial and the use of unfresh preparations should be avoided. In additions to that, it is also critical t...

Professional phagocytes, including macrophages, play a critical in the immune system. In addition to being the first line of defense in the innate immune system, they also play a critical role in activation of the adaptive immunity through their signaling and antigen presenting role1-3. While the function of professional phagocytes is multifaceted, phagosome maturation is the critical backbone of the bactericidal and antigen processing function of the professional phagocytes4,5. Upon the receptor mediated engulfment and uptake of a phagocytic target, such as bacteria, the nascent phagosome goes through a complex and well-orchestrated sequence of interaction and exchanges with compartments of the endocytic network and several other cellular organelles6. The nature of the compounds exchanged with these cellular entities as well as the regulation and the timing of fusion events determines the luminal phagosomal milieu and the phagosomal membrane composition and, thus7, the fate of the maturing phagosome8.
The physiological relevance of phagosome maturation is exemplified by the diverse strategies employed by various intracellular pathogens to escape from, arrest or subvert phagosome maturation9. Most of these strategies directly or indirectly prevent the final and critical stage of the phagosome maturation process: the fusion of phagosome with late endosomal/lysosomal compartments, which endows them with the bulk of the hydrolytic enzymes and anti-bacterial factors of a mature phagosome7,8,10. Analysis of this final and critical step therefore can provide us with strong indicators about the state of maturation of the phagosomes and if the natural physiological state is being positively or negatively affected under the particular experimental settings that are being utilized.
The late endosomal/lysosomal compartments are generally regarded to be the terminal compartments of the endocytic pathway, defined and distinguished from the early endocytic compartments by the presence of various, stage specific, marker molecules. For instance hydrolytic enzymes or membrane components such as Lysosomal associated membrane proteins (LAMPs) among others11. The terminal endocytic compartments -henceforth referred to simply as lysosomes- also serve as the main location for the final stages of digestion at the end of the phagocytic/endocytic pathway12. In this way, nondigestible probes can be loaded through endocytosis and macropinocytosis from the extracellular milieu and transported through the endocytic pathway to lysosomes, where it accumulates13,14&#160;after following a defined cycle of uptake and chase. Fluorophore-conjugated probes for fluorescent microscopy such as the organic nondigestible polymer dextran are commonly used as endocyticprobes15-17.
In this method, we describe the utilization of IgG-coated microbeads as phagocytic cargo to investigate phagosome maturation by analyzing the delivery of lysosomal luminal content to phagosomes. By using a fluorophore-conjugated dextran probe as a readily detectable marker of lysosomes in live bone marrow derived macrophages (BMM) and time-lapse video imaging with a confocal laser-scanning microscope, we follow the dynamic process of cargo delivery into phagosomes with high temporal resolution. We then describe how the collected time-lapse imaging data can be evaluated using the open-source and freely available image analysis software, Fiji (or ImageJ), statistically analyzed using Microsoft Excel and presented using GraphPad Prism software14,18.
Following the description of our method, analogous experiments can be designed using modified settings and factors to investigate their influence on phagosome maturation; virtually any other type of adherent primary or cell-line phagocytic cells, genetic loss of function experiments including gene knock-out and knock-down, mutants or expression of fusion proteins, phagocytic-targets, microbead coating compounds, endosomal/lysosomal probes and factors such a cytokines, chemical inhibitors, or siRNA&#160;among others are all variables that can be applied to the basic approach of this method.
We define the goal and the basic requirements of this method as follows:
<ul type="disc">
	<li>Goal of the method: to enable direct, quantitative and qualitative observation and analysis of phagosome maturation with high temporal resolution in living phagocytic cells.</li>
	<li>Phagocytic cargo: An appropriately coated microparticle or biological target. Here we use IgG-coated 3&#160;&#956;m spherical polystyrene microparticles that are readily internalized via the Fc-&#947; receptor mediated phagocytosis. Alternatively, any microorganism or coated microparticle for which a phagocytic receptor is present on the cells can be used.</li>
	<li>Phagocytic cells: Adherent phagocytic cells expressing the appropriate phagocytic receptors. Here we use mouse primary BMMs which abundantly express the Fc-&#947; receptors. Alternatively, dendritic cells (DC), monocytes or phagocytic epithelial cells or their genetic mutants or knock-out variants could be used.</li>
	<li>Lysosomal cargo: A fluorescently labeled lysosomal probe. Here, Texas Red-conjugated 70,000 kiloDaltons dextran (Dex70kD) is used as the luminal cargo of the lysosomes. Alternatively, any fluorescently detectable marker of a cellular compartment or organelle, or an appropriate probe for phagosomal properties such as acidity or degradative capacity, could be used to study the progression of phagosome maturation.</li>
	<li>Influencing factors: For instance, signaling molecules, chemical compounds, gene knock-down, or transient expression of mutant proteins could. Here, we have forgone the use of such a factor for the sake of simplicity, but for a recent example with mutant protein expression and knock-down influencing factors please see Kasmapour et al.18&#160;Any factor that can affect phagocytosis or the circumstances and mobility of phagosomes and/or the compartments that interact with maturing phagosomes could potentially be used.</li>
</ul>

<table border="0" cellpadding="0" cellspacing="0" width="610">
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagles Medium (D-MEM)</td>
			<td>PAA, Austria</td>
			<td>E15-009</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagles Medium without phenol red (D-MEM)</td>
			<td>PAA, Austria</td>
			<td>E15-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>PAA, Austria</td>
			<td>A15-151</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>PAA, Austria</td>
			<td>M11-004</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>PAA, Austria</td>
			<td>H15-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>PAA, Austria</td>
			<td>P11-010</td>
			<td></td>
		</tr>
		<tr>
			<td>WillCo-dish&#174; Glass bottom dish (35 mm &#8709;, #1.5)</td>
			<td>WillCo Wells, Netherlands</td>
			<td>GWSt-3522</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLviewTM glass bottom dish, 35 mm</td>
			<td>Greiner Bio one, Germany</td>
			<td>627871</td>
			<td>Alternative: Available as segmented</td>
		</tr>
		<tr>
			<td>Carboxylated latex microspheres</td>
			<td>Polysciences, USA</td>
			<td>#09850</td>
			<td>Available in different diameters, we used 3 &#956;m</td>
		</tr>
		<tr>
			<td>Polystyrene microparticles</td>
			<td>Kisker Biotech, Germany</td>
			<td>PPs-3.0COOH</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>ROTH, Germany</td>
			<td>305.1.3</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse whole IgG</td>
			<td>Rockland, USA</td>
			<td>010-0102</td>
			<td></td>
		</tr>
		<tr>
			<td>EDAC crosslinker</td>
			<td>Sigma-Aldrich, USA</td>
			<td>E6383-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mM Tris</td>
			<td>Sigma-Aldrich,USA</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>1-ml syringe Omnifix -F</td>
			<td>B.Braun, Germany</td>
			<td>9161406V</td>
			<td></td>
		</tr>
		<tr>
			<td>26&#160;G needle (0.45*25 mm)</td>
			<td>B.Baun, Germany</td>
			<td>4657683</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>PAA, Austria</td>
			<td>E15-039</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Gibco, USA</td>
			<td>16050-122</td>
			<td></td>
		</tr>
		<tr>
			<td>MES hydrate</td>
			<td>Sigma-Aldrich, USA</td>
			<td>M2933</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 &#956;M syringe mounted filter</td>
			<td>Sartorius, Germany</td>
			<td>83.1826.001</td>
			<td></td>
		</tr>
	</tbody>
</table>

study of phagolysosome biogenesis in live macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

The correct preparation of the cells and the beads for imaging is critical. Figure 1 shows the outline of the seeding, probe-loading and initial imaging steps in this method, based on our optimized protocol for BMMs. It is essential therefore that the protocol parameters be tested and optimized depending on the type of cells and probes that are to be used.
After addition of the IgG-coated beads to the preloaded cells, it is important that the field of view is chosen in a manne...

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Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

study-of-phagolysosome-biogenesis-in-live-macrophages

Research

JoVE Journal

Immunology and Infection

14.3K ビュー.  Helmholtz Centre for Infection Research. ライブマクロファージにおけるファゴリソソーム生合成の研究

ビデオ: Study of Phagolysosome Biogenesis in Live Macrophages

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in response to various immune stimuli. ROS generated during immune activation exerts potent antimicrobial activity primarily through the ability of ROS to damage DNA and proteins, causing death of microorganisms. Being able to measure ROS production reproducibly and with ease is necessary in order to assess the contribution of various pathways and molecules to this mechanism of host defense. In this paper, we demonstrate the use of fluorescent probes and flow cytometry to detect ROS production. Although widely used, fluorescent measurement of ROS is notoriously problematic, especially with regards to measurement of ROS induced by specific and not mitogenic stimuli. We present a detailed methodology to detect ROS generated as a result of specific Fc&#947;R stimulation beginning with macrophage generation, priming, staining, Fc&#947;R cross-linking, and ending with flow cytometric analysis.

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&amp;#947;R Cross-linking

This study demonstrates the use of flow cytometry to detect reactive oxygen species (ROS) production resulting from activation of the Fc&#947;R. This method can be used to assess changes in the antimicrobial and redox signaling function of phagocytes in response to immune complexes, opsonized microorganisms, or direct Fc&#947;R cross-linking.

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in ...