LPE is a Scottish Senior Clinical Fellow and acknowledges the support of the Chief Scientist Office (SCD/03). This work was funded by Wellcome Trust Project Grant to LPE (089930). NARG was funded by a Wellcome Trust Programme Grant (080088) and an equipment Grant (075470) (for DeltaVision), and by a FP7-2007-2013 Grant (HEALTH-F2-2010-260338-ALLFUN). We would like to thank the University of Aberdeen imaging facility, in particular Kevin MacKenzie, for helpful support and advice.

1. C. albicans Growth and Conditions
<ol>
	<li>Prepare SC-Ura agar plates by adding 6.9 g yeast nitrogen base without amino acids, 1 ml 1 M NaOH, 10 ml 1% (w/v) adenine hemisulphate salt and 20 g technical agar (previously described in detail in 11). Make up volume to 900 ml with distilled H2O. Autoclave, allow agar to cool, but not enough to solidify, and then add 50 ml sterile 40% D-glucose and 50 ml sterile 4% SC-Ura dropout under aseptic conditions. Mix, pour into Petri dishes and leave agar plates to cool and solidify. Store agar plates at 5 &deg;C until use.</li>
	<li>Streak C. albicans serotype A strain CAI4+CIp10 from glycerol stocks stored at -80 &deg;C onto SC-Ura agar plate.</li>
	<li>Incubate plate at 30 &deg;C until colonies form and store at 5 &deg;C.</li>
	<li>Culture a single C. albicans colony in 5 ml SC-Ura medium (recipe as in 1.1, but without technical agar) and incubate overnight at 30 &deg;C, 200 rpm to generate stationary phase C. albicans.</li>
</ol>
2. C. albicans Staining Using Fluorescein Isothiocyanate (FITC)
<ol>
	<li>Add 10 &mu;l C. albicans overnight culture to 990 &mu;l PBS (pH 7.4) and perform a cell count using a haemocytometer.</li>
	<li>To aid visualization of C. albicans during phagocytosis assays, stain 1 &times; 108 C. albicans using 1 mg/ml FITC in 0.05 M carbonate-bicarbonate buffer (pH 9.6) for 10 min at room temperature in the dark.</li>
	<li>To remove unbound FITC, wash C. albicans in 1 ml 1 x PBS, centrifuge at 3,000 &times; g for 5 min, remove supernatant and resuspend pellet in 1 ml 1 x PBS. Repeat 3 times.</li>
	<li>Resuspend pellet at 1 &times; 106 cells/&mu;l in 1 x PBS.</li>
</ol>
3. Preparation of the J774.1 Mouse Macrophage Cell Line
<ol>
	<li>Maintain J774.1 macrophages in 75 cm2 tissue culture flasks in DMEM medium supplemented with 10% (v/v) fetal calf serum (FCS), 200 U/ml penicillin/streptomycin and 2 mM L-glutamine at 37 &deg;C with 5% CO2. The preparation of primary macrophages is described elsewhere in detail 12, 13.</li>
	<li>Scrape J774.1 cells from the tissue culture flask and transfer to a 50 ml Falcon tube. Centrifuge at 600 &times; g for 5 min to obtain a cell pellet.</li>
	<li>Remove supernatant and resuspend pellet in 10 ml pre-warmed supplemented DMEM medium. Count cells using a haemocytometer and plate 1 &times; 106 J774.1 macrophages in 2 ml supplemented DMEM medium in a 35 mm glass-based imaging dish. Incubate overnight at 37 &deg;C, 5% CO2.</li>
	<li>Prior to imaging, replace supplemented DMEM medium with 2 ml pre-warmed supplemented CO2-independent medium (with 10% (v/v) fetal calf serum (FCS), 200 U/ml penicillin/streptomycin and 2 mM L-glutamine) containing 1 &mu;M LysoTracker Red DND-99.</li>
</ol>
4. Live Cell Video Microscopy Phagocytosis Assay
<ol>
	<li>The choice of microscope will depend upon what is available locally, but the microscope setup will need to include an inverted stage, an environmental chamber heated to 37 &deg;C and excitation/emission filters for the chosen stains (FITC and TRITC).</li>
	<li>Turn on the microscope heater prior to the experiment and allow sufficient time for the environmental control chamber to warm to 37 &deg;C. The time taken for chamber temperature to stabilize will vary for different microscope setups.</li>
	<li>Turn on the microscope and computer, and load the imaging software. Mount the imaging dish on the microscope stage and adjust the focus to find the J774.1 macrophages. Optimize the appearance of TRITC and DIC images by adjusting the percentage of transmitted light and exposure times.</li>
	<li>Remove the imaging dish and add 3 &times; 106 FITC-stained C. albicans to the dish. Record the time that C. albicans is added to the dish. Return the dish to the stage and optimize the appearance of FITC images if necessary. Set up a points list if required.</li>
	<li>Commence imaging when all points are in focus and the channels are optimized. Capture FITC, TRITC and DIC images every minute for 6 hr.</li>
</ol>

<ol>
	<li>McKenzie, C. G. J., 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=Contribution+of+Candida+albicans+cell+wall+components+to+recognition+by+and+escape+from+murine+macrophages.">Contribution of Candida albicans cell wall components to recognition by and escape from murine macrophages.</a> Infect. Immun. 78, 1650-1658 (2010).</li><li>Mora-Montes, H. M., 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=Recognition+and+blocking+of+innate+immunity+cells+by+Candida+albicans+chitin.">Recognition and blocking of innate immunity cells by Candida albicans chitin.</a> Infect. Immun. 79, 1961-1970 (2011).</li><li>Keppler-Ross, S., 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=Recognition+of+yeast+by+murine+macrophages+requires+mannan+but+not+glucan.">Recognition of yeast by murine macrophages requires mannan but not glucan.</a> Eukaryot. Cell. 9, 1776-1787 (2010).</li><li>Vijayan, D., 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=Mincle+polarizes+human+monocyte+and+neutrophil+responses+to+Candida+albicans.">Mincle polarizes human monocyte and neutrophil responses to Candida albicans.</a> Immunol. Cell Biol. , (2012).</li><li>Seider, K., 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=The+facultative+intracellular+pathogen+Candida+glabrata+subverts+macrophage+cytokine+production+and+phagolysosome+maturation.">The facultative intracellular pathogen Candida glabrata subverts macrophage cytokine production and phagolysosome maturation.</a> J. Immunol. 187, 3072-3086 (2011).</li><li>Sheth, C., 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=Glycosylation+status+of+the+C.+albicans+cell+wall+affects+the+efficiency+of+neutrophil+phagocytosis+and+killing+but+not+cytokine+signalling.">Glycosylation status of the C. albicans cell wall affects the efficiency of neutrophil phagocytosis and killing but not cytokine signalling.</a> Med. Mycol. (5), 513-524 (2011).</li><li>Lewis, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage+specific+assessment+of+Candida+albicans+phagocytosis+by+macrophages+identifies+cell+wall+composition+and+morphogenesis+as+key+determinants.">Stage specific assessment of Candida albicans phagocytosis by macrophages identifies cell wall composition and morphogenesis as key determinants.</a> PLoS Pathog. 8 (3), e1002578 (2012).</li><li>Klaas, M., 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=Sialoadhesin+Promotes+Rapid+Proinflammatory+and+Type+I+Interferon+Responses+to+a+Sialylated+Pathogen,+Campylobacter+jejuni.">Sialoadhesin Promotes Rapid Proinflammatory and Type I Interferon Responses to a Sialylated Pathogen, Campylobacter jejuni.</a> J. Immunol. , (2012).</li><li>Lewis, L. 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=Candida+albicans+infection+inhibits+macrophage+cell+division+and+proliferation.">Candida albicans infection inhibits macrophage cell division and proliferation.</a> Fungal Genet. Biol. , (2012).</li><li>Bain, J. M., 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=Non-lytic+expulsion/exocytosis+of+Candida+albicans+from+macrophages.">Non-lytic expulsion/exocytosis of Candida albicans from macrophages.</a> Fungal Genet. Biol. , (2012).</li><li>Mora-Montes, H., 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=Interactions+between+macrophages+and+cell+wall+oligosaccharides+of+Candida+albicans.">Interactions between macrophages and cell wall oligosaccharides of Candida albicans.</a> Methods Mol. Biol. 845, 247-260 (2012).</li><li>McPhillips, K., 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=Assessment+of+apoptotic+cell+phagocytosis+by+macrophages.">Assessment of apoptotic cell phagocytosis by macrophages.</a> Methods Mol. Biol. 559, 247-256 (2009).</li><li>Erwig, L. -. P., 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=Differential+regulation+of+phagosome+maturation+in+macrophages+and+dendritic+cells+mediated+by+Rho+GTPases+and+ezrin-radixin-moesin+(ERM)+proteins.">Differential regulation of phagosome maturation in macrophages and dendritic cells mediated by Rho GTPases and ezrin-radixin-moesin (ERM) proteins.</a> Proc. Natl. Acad. Sci. U.S.A. 103 (34), 12825-12830 (2006).</li></ol>

The authors have nothing to disclose. NG is a recipient of unrestricted grant support for basic research by Gilead Sciences. LE works as a consultant for GSK in early drug development.

Here the method for the use of live-cell video microscopy to study macrophage phagocytosis is described. Video microscopy offers multiple additional layers of information for analysis. One basic advantage is that uptake data can be generated (from a single experiment) for any time point throughout the 6 hr observation period. More importantly, the method described enables differential analysis of the individual stages of phagocytosis. We have, for example, shown that changes in overall uptake of C. albicans glycosylation and morphogenesis mutants by macrophage cell lines and primary macrophages can be either a consequence of changes in macrophage migration towards target cells or rate of engulfment once cell-cell contact is established 7.
There are a number of pitfalls to avoid when conducting these experiments. First, it is very important to ensure stable environmental conditions throughout the experimental procedure. This is best achieved in an environmental chamber that is set to the experimental conditions several hours before commencing the experiment. Video quality is highly dependent on sophisticated modules that use infrared lasers to automatically maintain the sample z-position regardless of mechanical or thermal changes thus eliminating the need for manual focus corrections.
Given the extended nature of the experiments, it is important to limit laser light exposure and associated photobleaching and photoconversion effects, which can be minimized by employing highly fluorescent markers, which facilitate low exposure times. The FITC staining protocol described here is quick and reliable. As FITC is a very bright and stable stain, low exposure times are required and this makes FITC ideal for extended time-lapse movies. However, we routinely use a variety of different target stains, including Calcofluor White and PKH dyes as well as tagged organisms.
Most of our published work is conducted using a wide-field microscope, but exposure can be further minimized by using a spinning disc confocal microscope and in our hands this is essential for time-lapse 3D video microscopy.
During this study, images were captured at 1 min intervals over a 6 hr phagocytosis assay. The interval between images can be adjusted depending on the process under investigation. For example, the interval can be decreased when investigating rapid processes such as vesicular trafficking. The minimum time interval will be limited by microscope and camera specifications. There are some considerations to bear in mind when adjusting timings. First, decreasing the interval between snapshots will mean fewer points can be imaged and file size will be considerably increased. On the contrary, increasing the image interval too much will make the movie lose continuity.
This approach can be applied in principle to study other pathogens and uptake of dying host cells. For example, we have recently shown that bone-marrow derived macrophages from sialoadhesin-deficient mice exhibit greatly reduced binding and phagocytosis of sialylated Campylobacter jejuni 8. However, target size is an important determinant for feasibility, as image analysis becomes increasingly difficult with a reduction in target cell size. Sophisticated image analysis software is essential for rapid video microscopy analysis, and this should be coupled with appropriate bioinformatics support to facilitate the generation of algorithms to study migration of individual cells and entire cell populations 7. Live-cell video microscopy in combination with sophisticated image analysis software for the minute-by-minute analysis of migration and individual macrophage-C.albicans interactions, provides unique insight into the complexity of C. albicans phagocytosis by macrophages. There is enormous potential to expand these methods to study other pathogens and phagocytes (dendritic cells, neutrophils) and to develop 3D video microscopy to image cell-cell interactions in greater detail or on more physiological surfaces such as epithelial and endothelial cell layers. This technique is part of the next generation of tools in the study of host-pathogen interactions and will help generate detailed spatial and temporal information on a broad range of dynamic processes.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Yeast nitrogen base without amino acids</td>
 <td>Formedium</td>
 <td>CYN0402</td>
 </tr>
 <tr>
 <td>NaOH</td>
 <td>Sigma</td>
 <td>S5881-500G</td>
 </tr>
 <tr>
 <td>Adenine hemisulphate salt</td>
 <td>Sigma</td>
 <td>A3159-25G</td>
 </tr>
 <tr>
 <td>Agar technical (no. 3)</td>
 <td>Oxoid</td>
 <td>LP0013</td>
 </tr>
 <tr>
 <td>D-glucose</td>
 <td>Sigma</td>
 <td>G7021-1KG</td>
 </tr>
 <tr>
 <td>SC-Ura dropout</td>
 <td>Formedium</td>
 <td>DSCK102</td>
 </tr>
 <tr>
 <td>FITC</td>
 <td>Sigma</td>
 <td>F7250-1G</td>
 </tr>
 <tr>
 <td>Sodium carbonate</td>
 <td>BDH</td>
 <td>301215M</td>
 </tr>
 <tr>
 <td>Sodium bicarbonate</td>
 <td>BDH</td>
 <td>102475W</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Gibco</td>
 <td>18912-014</td>
 </tr>
 <tr>
 <td>DMEM </td>
 <td>Lonza</td>
 <td>BE12-614F</td>
 </tr>
 <tr>
 <td>FCS</td>
 <td>Life Technologies</td>
 <td>10106169</td>
 </tr>
 <tr>
 <td>Penicillin/streptomycin </td>
 <td>PAA</td>
 <td>P11-010</td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>Lonza</td>
 <td>BE17-605E</td>
 </tr>
 <tr>
 <td>LysoTracker Red DND-99</td>
 <td>Invitrogen</td>
 <td>L7528</td>
 </tr>
 <tr>
 <td>35 mm glass-dishes</td>
 <td>PAA</td>
 <td>PAA12305160X</td>
 </tr>
 </tbody>
</table>

live-cell video microscopy of fungal pathogen phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

Here we show representative results for C. albicans uptake by a murine macrophage cell line J774.1 during a 6 hr live-cell video microscopy experiment. Figure 1 is a representative live-cell video microscopy movie, showing phagocytosis of C. albicans by murine J774.1 macrophages. Live-cell video microscopy enables internalization of C. albicans to be visualized without the need to stain external Candida. However, during these experiments, C. albicans was stained using the pH-sensitive dye FITC (green), which is quenched during acidification of the macrophage phagosome, and facilitates confirmation that Candida has been internalized (Figure 1). To further aid visualization of ingested C. albicans, macrophages were stained using the red fluorescent dye LysoTracker red DND-99, which stains acidic compartments and serves as a non-specific marker of phagosome maturation. Figure 2 is a still image from a live-cell video microscopy experiment and illustrates the variation in the number of C. albicans ingested by individual J774.1 macrophages. In this experiment, 82% of J774.1 macrophages engulfed at least one C. albicans cell at the end of the 6 hr phagocytosis assay. In Figure 3, the number of internalized C. albicans cells per macrophage is reported. The mean number of C. albicans taken up per macrophage is 3.4, but interestingly macrophages can ingest up to 16 fungal cells. Figure 4 is a sequence of still images from a representative live-cell video microscopy experiment showing a macrophage phagocytosing C. albicans cells, hypha growth with the macrophage and ultimately macrophage lysis. Figure 4 illustrates that video microscopy enables analysis of events following engulfment of target cells, i.e. data can be generated for the killing of macrophages by C. albicans hyphae in relation to the number and morphogenesis of ingested target cells, and how macrophage killing relates to phagosome maturation which can be studied in real time.
Figure 1. Live-cell video microscopy movie showing phagocytosis of C. albicans by murine J774.1 macrophages. Macrophages are stained using the red fluorescent dye LysoTracker red DND-99, and C. albicans are stained using FITC (green). Macrophages and C. albicans were co-cultured for a period of 6 hr at 37 &deg;C in complete CO2-independent medium. Images were captured at 1 min intervals for 6 hr. Macrophages can be seen establishing cell-cell contact with C. albicans, which is then followed by uptake of C. albicans into macrophage phagosomes. Arrows point to C. albicans prior to engulfment by J774.1 macrophages. This video also shows that FITC fluorescence is quenched following engulfment of C. albicans. Scale bar, 10 &mu;m. <a href="https://www.jove.com/files/ftp_upload/50196/50196fig1.avi" target="_blank"> Click here to view movie</a>.
<img alt="Figure 2" src="/files/ftp_upload/50196/50196fig2.jpg" /> 
 Figure 2. Representative live-cell video microscopy still image showing variation in the number of C.albicans engulfed by individual macrophages. Macrophages (stained using LysoTracker red DND-99) were co-cultured with FITC-stained C. albicans (green). There is variation in the number of C. albicans engulfed (a number next to an arrow indicates the number of C. albicans engulfed), and in C. albicans morphology (i.e. yeast verses hypha). Scale bar, 20 &mu;m.
<img alt="Figure 3" src="/files/ftp_upload/50196/50196fig3.jpg" /> 
 Figure 3. Graph showing the number of C. albicans ingested per J774.1 murine macrophage at the end of 6 hr phagocytosis assays. The data represent 2 independent experiments. A total of 100 macrophages were counted from 3 fields from each experiment, giving a total of 600 individual macrophages.
<img alt="Figure 4" src="/files/ftp_upload/50196/50196fig4.jpg" /> 
 Figure 4. A series of images from a representative live-cell video microscopy experiment showing a macrophage (M) and C. albicans (C) prior to and during recognition (A, B), and during and after uptake (C, D). C. albicans hyphae continue to grow within the macrophage (E, F), which can result in macrophage lysis (G). Other macrophages are recruited to the site of rupture and attempt to ingest the released C. albicans (H). Macrophages are stained using the red fluorescent dye LysoTracker red DND-99, and C. albicans are stained using FITC (green). Note that FITC staining is quenched following C. albicans uptake by J774.1 macrophages (C). Scale bar, 10 &mu;m.

Watch this Scientific Journal Video about Live-cell Video Microscopy of Fungal Pathogen Phagocytosis at JoVE.com

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

live-cell-video-microscopy-of-fungal-pathogen-phagocytosis

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Immunology and Infection

38.8K Views.  University of Aberdeen. We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Video: Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

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 ...

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"1. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles2-4.
The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.
The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania5, Listeria6, Mycobacterium7, Rhodococcus8, Salmonella9 or Legionella spp.10. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.
The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation12,13 (Fig. 1).
A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before13. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread.

Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Here, we present methodologies that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons.

Advances in live cell fluorescence microscopy  techniques, as well as the construction of recombinant viral strains that  express fluorescent fusion ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.