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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The authors have nothing to disclose.

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 ...

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Research

JoVE Journal

Immunology and Infection

14.3K visualizações.  Helmholtz Centre for Infection Research. Estudo da fagolisossomo Biogenesis em macrófagos ao vivo