We thank Mrs. Pia Stier for excellent technical assistance. M. von Lilienfeld-Toal is supported by the Center for Sepsis Control and Care (German Federal Ministry of Education and Health, BMBF, FKZ 01E01002) and InfectoGnostics Research Campus (BMBF, FKZ 13GW0096D). Thi Ngoc Mai Hoang is supported by the Jena School of Microbial Communication (Deutsche Forschungsgemeinschaft, FKZ 214/2)

This protocol includes the use of human buffy coats obtained from the Institute for Transfusion Medicine, Jena University Hospital and fresh venous blood drawn from patients, both after written informed consent of the donors in accordance to ethics committee approval 4357-03/15.
1. Preparation of Aspergillus fumigatus Conidia
<ol>
	<li>Grow A. fumigatus on 1.5% malt agar Petri dishes for 5 days at 37 &#176;C without CO2. 
	CAUTION: A. fumigatus is a biosafety level 2 microorganism and must be handled in an appropriate facility using a biosafety cabinet and wearing lab coat, gloves and a filter mask. 
	NOTE: The plate should be covered completely with a green-greyish layer of conidia. White cultures do not sporulate. Composition of malt agar: 4% (w/v) malt extract, yeast extract 0.4% (w/v), agar 1.5% (w/v), Aqua dest.</li>
	<li>Harvesting conidia
	<ol>
		<li>Place a paper towel wet with disinfectant into the biosafety cabinet and put the plate on top to prevent overdistribution of volatile conidia.</li>
		<li>Add 10 mL of phosphate buffered saline (PBS) + 0.01% detergent on top of the fungus, use a Drigalski spatula to spread the liquid over the plate and rub off the dark colored conidia. Be careful not to remove the white mycelium.</li>
		<li>Put the conidia suspension to a 50 mL tube using a 30 &#181;m cell strainer to remove any residual mycelium.</li>
		<li>Repeat steps 1.2.2 and 1.2.3 and collect in the same 50 mL tube.</li>
		<li>Spin for 5 min at 2,600 x g at room temperature.</li>
		<li>Remove the supernatant and resuspend in 20 mL of sterile Aqua dest.</li>
		<li>Determine conidia concentration with a Thoma counting chamber. 
		NOTE: The protocol can be paused here and the conidia suspension stored for up to 1 month at 4 &#176;C with the lid screwed tightly.</li>
	</ol>
	</li>
	<li>FITC labeling
	<ol>
		<li>Prepare a 0.1 mM solution of FITC powder in sterile 0.1 M Na2CO3 (dissolved in PBS). 
		CAUTION: FITC powder is hazardous and should be handled with gloves, goggles and a filter mask. Collect waste according to local regulations. 
		NOTE: Omit artificial light during this and the following steps involving conidia.</li>
		<li>Resuspend 1 x 108 (or less) conidia in 5 mL of FITC solution in a 15 mL tube. Incubate for 20 min at 37 &#176;C in a rotator.</li>
		<li>For the negative control, resuspend conidia in 0.1 M Na2CO3 (without FITC) in a 15 mL tube. Incubate for 20 min at 37 &#176;C in a rotator. 
		NOTE: When calculating the amount of conidia to be stained, take into account a loss of up to 70% during staining, swelling and all necessary washing steps. If no rotator is available, the suspension should be shaken three times during incubation.</li>
		<li>For washing, add 10 mL of PBS + 0.01% detergent to the suspension and spin for 5 min at 2,600 x g at room temperature.</li>
		<li>Remove supernatant and repeat washing twice with 10 mL of PBS + 0.01% detergent.</li>
	</ol>
	</li>
	<li>Swelling of conidia (may be omitted if not desired)
	<ol>
		<li>Resuspend FITC labeled conidia in 5 mL of Roswell Park Memorial Institute (RPMI) medium + 10% Fetal Calf Serum (FCS) and incubate in a rotator at 37 &#176;C for the desired time (e.g., 2 h, 4 h). 
		NOTE: If no rotator is available, the suspension should be shaken at least every 20 min during incubation.</li>
		<li>Add 10 mL of PBS + 0.01% detergent to the suspension and spin for 5 min at 2,600 x g at room temperature.</li>
		<li>Remove the supernatant and wash twice with 10 mL of PBS + 0.01% detergent.</li>
		<li>Filter through a 30 &#181;m cell strainer to remove large clumps of conidia.</li>
	</ol>
	</li>
	<li>Fixing of conidia (may be omitted if not desired)
	<ol>
		<li>Resuspend FITC labeled and swollen conidia in 1 mL of formaldehyde and incubate for 1 h at room temperature.</li>
		<li>Add 10 mL of PBS + 0.01% detergent to the suspension and spin for 5 min at 2,600 x g at room temperature.</li>
		<li>Remove the supernatant and wash twice with 10 mL of PBS + 0.01% detergent. 
		NOTE: The protocol can be paused here and the conidia stored in PBS in the dark (tube wrapped up in aluminum foil) for up to 1 week at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Preparation of Human Primary Leukocytes
<ol>
	<li>Put 5 mL of buffy coat in a 50 mL tube. Alternatively, use fresh human peripheral venous blood drawn into ethylenediaminetetraacetic acid (EDTA) monovettes (10 mL per 50 mL tube). 
	CAUTION: Human blood may transmit viruses such as Human Immunodeficiency Virus and Hepatitis B Virus. Handle only after being vaccinated against Hepatitis B. Handle in a biosafety cabinet wearing lab coat and gloves.</li>
	<li>Fill up the tube with Erythrocyte Lysis (EL) Buffer, invert three times and incubate for 5-8 min horizontally until the milky appearance of the mixture turns clear. 
	NOTE: Occasionally, the blood may take longer to lyse. Go by the appearance. It is not unusual that two tubes of the same blood take different times to lyse. Composition of EL buffer: 0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA</li>
	<li>Spin for 10 min at 300 x g at room temperature. Discard the supernatant.</li>
	<li>Resuspend the pellet in 1 mL of EL Buffer by pipetting. Then add another 24 mL of EL buffer, and invert several times.</li>
	<li>Spin for 5 min at 300 x g at room temperature.</li>
	<li>Discard the supernatant and resuspend cells in 1 mL of RPMI + 10% FCS.</li>
	<li>Determine the cell concentration with a Neubauer counting chamber.</li>
</ol>
3. Phagocytosis Assay
<ol>
	<li>Incubate 2 x 106 leukocytes and 4 x 106 FITC-labeled conidia (multiplicity of infection = 2) in 1.5 mL of RPMI + 10% FCS in a 12-well cell culture plate. As controls, include cells only (no conidia) and cells + unlabeled conidia.</li>
	<li>Place in a humidified CO2 incubator at 37 &#176;C and incubate for the desired period of time (e.g., 0.5 h, 2 h or 4 h).</li>
	<li>After incubation, harvest cells with a cell scraper and put in a 15 mL tube.</li>
	<li>Spin for 5 min at 300 x g at room temperature.</li>
	<li>Collect supernatant for cytokine analysis or discard if not wanted. Resuspend each sample in 100 &#181;L of PBS + 2 mM EDTA.</li>
</ol>
4. Antibody Staining
<ol>
	<li>For each sample, prepare 100 &#181;L of antibody mix, including APC anti-FITC antibody, according to Table 1.</li>
	<li>Put a 100 &#181;L sample into one well of a 96-well V-bottom plate. Add 150 &#181;L of PBS + 2 mM EDTA for washing.</li>
	<li>For color compensation, place 1 x 106 cells for each color in further wells of the 96-well V bottom plate. Include a well of cells that is left unstained. Add 150 &#181;L of PBS + 2 mM EDTA for washing.</li>
	<li>Cover plate with an adhesive foil.</li>
	<li>Spin for 5 min at 300 x g at room temperature. Remove the foil.</li>
	<li>Discard the supernatant by quickly and forcefully inverting the plate only once over the sink or a disposable paper towel. 
	NOTE: Do not repeat or knock the plate on a paper until dry as this will result in a massive loss of cells from the plate.</li>
	<li>Resuspend cells in the 100 &#181;L antibody mix, and mix well by pipetting.</li>
	<li>For color compensation, resuspend the respective cells in 100 &#181;L of PBS + 2 mM EDTA and add a single antibody to each well at the same amount used in the antibody mix.</li>
	<li>Cover with an adhesive foil and incubate for 20 min at room temperature in the dark.</li>
	<li>Remove the foil. Add 150 &#181;L of PBS + 2 mM EDTA to each well for washing. Cover with an adhesive foil.</li>
	<li>Spin for 5 min at 300 x g at room temperature. Remove the foil. Discard the supernatant by quickly and forcefully inverting the plate over the sink or a disposable paper towel.</li>
	<li>Resuspend in 200 &#181;L of PBS + 2 mM EDTA and transfer cells from each well to a separate round bottom tube. Make sure there are no cell clusters in the suspension. Remove clusters otherwise. 
	NOTE: Every cluster that is large enough for the eye to see is large enough to potentially clog the cytometer.</li>
</ol>
5. Flow Cytometry
<ol>
	<li>Start the flow cytometer and let it warm up. Start the acquisition software.</li>
	<li>Create a new experiment and setup and label the samples.</li>
	<li>Set up parameters (FSC 250, SSC 250) and detectors for fluorophores FITC, APC, BUV395, V500, PerCP-Cy5.5.</li>
	<li>Compensation setup
	<ol>
		<li>Open compensation setup.</li>
		<li>Indicate individual colors.</li>
		<li>Using the control cells left unstained or with individual stainings, set the PMT detector voltages to include all events within the scale.</li>
		<li>Record at least 10,000 events of each control.</li>
		<li>Use the compensation setup to calculate the spillover of fluorophores and apply to the experiment&#39;s cytometer settings.</li>
	</ol>
	</li>
	<li>Recording sample data
	<ol>
		<li>Display FSC and SSC in the acquisition software and set gate around leukocytes.</li>
		<li>Based on the leukocyte gate, display dot plot SSC/CD45 and gate for CD45+ cells to separate from conidia.</li>
		<li>Display CD45+ cells in a dot plot CD14/CD66b and gate monocytes (CD14+) and neutrophils (CD66b+) separately.</li>
		<li>Display neutrophils in a dot plot anti-FITC/FITC.</li>
		<li>Using the sample with unlabeled conidia, set quadrants for anti-FITC and FITC signals, allowing a maximum of 1% of cells in the respective quadrants.</li>
		<li>Repeat steps 5.5.4 and 5.5.5 for monocyte gate.</li>
		<li>Record all samples with at least 20,000 events in the leukocyte gate.</li>
	</ol>
	</li>
</ol>

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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=A+novel+assay+of+antimycobacterial+activity+and+phagocytosis+by+human+neutrophils.">A novel assay of antimycobacterial activity and phagocytosis by human neutrophils.</a> Tuberculosis (Edinb). 93 (2), 167-178 (2013).</li><li>Nuutila, J., Lilius, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+quantitative+determination+of+ingestion+by+phagocytes+needs+the+distinguishing+of+overlapping+populations+of+binding+and+ingesting+cells.">Flow cytometric quantitative determination of ingestion by phagocytes needs the distinguishing of overlapping populations of binding and ingesting cells.</a> Cytometry A. 65 (2), 93-102 (2005).</li><li>Saresella, 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=A+rapid+evaluation+of+phagocytosis+and+killing+of+Candida+albicans+by+CD13++leukocytes.">A rapid evaluation of phagocytosis and killing of Candida albicans by CD13+ leukocytes.</a> Journal of Immunological Methods. 210 (2), 227-234 (1997).</li><li>Fattorossi, A., Nisini, R., Pizzolo, J. 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The authors have nothing to disclose.

This protocol presents a fast flow cytometric method to measure interaction of A. fumigatus conidia with a large number of primary human leukocytes that is not possible in common microscopic protocols. Imaging cells with a microscope and manually counting internalized conidia is cumbersome and can realistically be done for a few hundred cells only. Flow cytometry overcomes this problem by measuring thousands of cells within minutes. A hurdle common to both approaches is the distinction of phagocytosed and adhere...

Invasive pulmonary aspergillosis is a great threat to immunocompromised patients as treatment options are limited and only successful upon early diagnosis, which leads to high mortality rates1. Infectious agents are conidia (spores) of the mold Aspergillus fumigatus that are ubiquitous to most habitats2. Conidia are inhaled, pass through the airways and can finally enter the lung alveoli. In immunocompetent humans, these conidia are cleared by innate immune cells such as monocytes or macrophages and neutrophil granulocytes, which take up (phagocytose) and digest the pathogens3. Phagocytosis is important for microbiologists and immunologists likewise when interested in host-pathogen interactions. Confrontation assays, such as co-incubation of leukocytes and conidia, often include labeling of the spores by fluorescein or its derivative fluorescein isothiocyanate (FITC). Using a microscope, it is straightforward to identify internalized fluorescent conidia and to determine attached/adherent conidia, although this approach is cumbersome and realistically restricted to a few hundreds of cells4. However, in flow cytometry which easily allows the analysis of hundreds of thousands of cells within minutes, differential staining of phagocytosed and adherent conidia is vital. Therefore, many protocols rely on Trypan Blue to quench FITC-fluorescence from adherent conidia5,6,7,8. Another approach is exploiting fluorescence resonance energy transfer of ethidium bromide and FITC to emit red instead of green fluorescence from adherent conidia9,10,11. If specific antibodies are available, as is the case for some bacteria, cell-bound particles can be directly stained12,13.
Here, we present a protocol to quickly and quantitatively assess phagocytosis of FITC-labeled A. fumigatus conidia by human leukocytes along with attachment of spores to cells and lack of interaction by employing an allophycocyanin (APC)-coupled anti-FITC antibody. The method also allows for the simultaneous flow cytometric analysis of further cell markers that can be employed for separate analysis of phagocytosis by monocytes and neutrophils from the same sample.
The protocol can be applied for characterization of fungal strains (e.g., several species of Aspergillus and other molds from the genus Mucorales presented here) and their mutants14 and immunological research on phagocytes, such as leukocytes from immunocompromised individuals.

<table><tbody><tr><td>Adhesive foil</td><td>Brand</td><td>701367</td><td></td></tr><tr><td>anti-CD14 V500</td><td>BD Biosciences</td><td>561391</td><td>clone M5E2</td></tr><tr><td>anti-CD45 BUV395</td><td>BD Biosciences</td><td>563792</td><td>clone HI30</td></tr><tr><td>anti-CD66b PerCP-Cy5.5</td><td>BD Biosciences</td><td>562254</td><td>clone G10F5</td></tr><tr><td>anti-FITC APC</td><td>ThermoFisher Scientific</td><td>17-7691-82</td><td>clone NAWESLEE</td></tr><tr><td>Cell culture plate, 12-well</td><td>Greiner Bio-one</td><td>665180</td><td></td></tr><tr><td>Cell scraper</td><td>Bioswisstech</td><td>800020</td><td></td></tr><tr><td>Cell strainer, 30 &amp;micro;m</td><td>Miltenyi Biotech</td><td>130-098-458</td><td>SmartStrainer</td></tr><tr><td>Cytometer</td><td>BD Biosciences</td><td></td><td>LSR Fortessa II, lasers: 488 nm (blue), 405 nm (violet), 355 nm (UV) and 640 nm (red)</td></tr><tr><td>Detergent</td><td>Sigma Aldrich</td><td>P1379</td><td>Tween 20, 0.01% in PBS</td></tr><tr><td>Drigalski spatula</td><td>Carl Roth</td><td>PC59.1</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma Aldrich</td><td>ED3SS-500g</td><td>2 mM in PBS</td></tr><tr><td>Erythrocyte lysis buffer</td><td></td><td></td><td>0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA</td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>Biochrom AG</td><td>S 0115</td><td>10% in RPMI 1640</td></tr><tr><td>Fluorescein isothiocyanate (FITC)</td><td>Sigma Aldrich</td><td>F3651-100MG</td><td>0.1 mM in Na2CO3 /PBS solution</td></tr><tr><td>Formaldehyd</td><td>Carl Roth</td><td>PO87.3</td><td>Histofix</td></tr><tr><td>Malt agar (1.5%)</td><td></td><td></td><td>malt extract (40 g), yeast extract (4 g), agar (15 g), Aqua dest. (1 L), adjust pH to 5.7-6.0, sterilise at 121 &amp;deg;C for 35 minutes</td></tr><tr><td>Na2CO3</td><td>Carl Roth</td><td>8563.1</td><td>0.1 M in PBS</td></tr><tr><td>Petri dish</td><td>Greiner Bio-one</td><td>633180</td><td></td></tr><tr><td>Phosphat Buffered Saline (PBS)</td><td>ThermoFisher Scientific</td><td>189012-014</td><td>without Calcium, without Magnesium</td></tr><tr><td>RPMI 1640</td><td>ThermoFisher Scientific</td><td>61870010</td><td>RPMI 1640 Medium, GlutaMAX Supplement</td></tr><tr><td>Rotator</td><td>Miltenyi Biotech</td><td>130-090-753</td><td>MACSmix Tube Rotator</td></tr><tr><td>Round-bottom tube, 7.5 mL</td><td>Corning</td><td>REF 352008</td><td></td></tr><tr><td>Software for data acquisition and analysis</td><td>BD Biosciences</td><td></td><td>FACSDiva 8.0</td></tr><tr><td>V-bottom plate, 96 well</td><td>Brand</td><td>781601</td><td>untreated surface</td></tr></tbody></table>

measuring phagocytosis of aspergillus fumigatus conidia by human leukocytes using flow cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

In measuring phagocytosis of A. fumigatus conidia by human phagocytic cells, discrimination between genuine internalization and mere attachment of conidia to the cells is an obstacle, especially when it comes to high-throughput methods such as flow cytometry. In order to overcome this hurdle, we present a fast and reliable protocol based on the staining of conidia with the fluorescent dye FITC prior to co-incubation of cells and conidia, followed by a counterstaining with an APC-labeled anti-FITC antibody after ...

Watch this Scientific Journal Video about Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry at JoVE.com

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...

measuring-phagocytosis-aspergillus-fumigatus-conidia-human-leukocytes

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Immunology and Infection

6.7K Views.  Leibniz Institute for Infection Biology and Natural Product Research. This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

Video: Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells.

Here, we demonstrate how image cytometry can be used for quantification of pathogenic fungi in association with host cells in culture. This technique can be used as an alternative to CFU enumeration.

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans ...

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 ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse models of pulmonary fungal infection. Although multiple methods are used to assess fungal burden, quantitative polymerase chain reaction (qPCR) of fungal DNA has emerged as a technique with several advantages over previous culture-based methods. Currently, a comprehensive assessment of lung pathology, leukocyte recruitment, fungal burden, and gene expression in mice with invasive aspergillosis (IA) necessitates the use of a significant number of experimental and control animals. Here the quantification of lung histological staining to determine fungal burden using a reduced number of animals was examined in detail. Lung sections were stained to identify fungal structures with Gomori&#39;s modified methanamine silver (GMS) staining. Images were taken from the GMS-stained sections from 4 discrete fields of each formalin-fixed paraffin-embedded lung. The GMS stained areas within each image were quantified using an image analysis program, and from this quantification, the mean percentage of stained area was determined for each sample. Using this strategy, eosinophil-deficient mice exhibited decreased fungal burden and disease with caspofungin therapy, while wild-type mice with IA did not improve with caspofungin. Similarly, fungal burden in mice lacking &#947;&#948; T cells were also improved by caspofungin, as measured by qPCR and GMS quantification. GMS quantification is therefore introduced as a method for the determination of relative lung fungal burden that may ultimately reduce the quantity of experimental animals required for comprehensive studies of invasive aspergillosis.

Here we describe a protocol to determine pulmonary fungal burden in mice with invasive aspergillosis by quantification of Gomori&#39;s modified methanamine silver staining in histological sections. Use of this method resulted in comparable results with less animals compared to assessment of fungal burden by quantitative PCR of lung fungal DNA.

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse ...

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung

Aspergillus fumigatus conidia are airborne pathogens that can penetrate human airways. Immunocompetent people without allergies exhibit resistance and immunological tolerance, while in immunocompromised patients, conidia can colonize airways and cause severe invasive respiratory disorders. Various cells in different airway compartments are involved in the immune response that prevents fungal invasion; however, the spatio-temporal aspects of pathogen elimination are still not completely understood. Three-dimensional (3D) imaging of optically cleared whole-mount organs, particularly the lungs of experimental mice, permits detection of fluorescently labeled pathogens in the airways at different time points after infection. In the present study, we describe an experimental setup to perform a quantitative analysis of A. fumigatus conidia distribution in the airways. Using fluorescent confocal laser scanning microscopy (CLSM), we traced the location of fluorescently labeled&#160;conidia in the bronchial branches and the alveolar compartment 6 hours after oropharyngeal application to mice. The approach described here was previously used for detection of the precise pathogen location and identification of the pathogen-interacting cells at different phases of the immune response. The experimental setup can be used to estimate the kinetics of the pathogen elimination in different pathological conditions.

Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung

We describe the method for quantitative analysis of the distribution of Aspergillus fumigatus conidia (3 &#181;m in size) in the airways of mice. The method also can be used for the analysis of microparticles and nanoparticle agglomerate distribution in the airways in various pathological condition models.

Aspergillus fumigatus conidia are airborne pathogens that can penetrate human airways. Immunocompetent people without allergies exhibit resistance and ...

Fungal Clearance Efficiency of Macrophages

Viability Assay of Trichoderma stromaticum Conidia Inside Human Peripheral Blood Mononuclear-Derived Macrophages

Macrophages represent a crucial line of defense and are responsible for preventing the growth and colonization of pathogens in different tissues. Conidial phagocytosis is a key process that allows for the investigation of the cytoplasmic and molecular events involved in macrophage-pathogen interactions, as well as for the determination of the time of death of internalized conidia. The technique involving the phagocytosis of fungal conidia by macrophages is widely used for studies evaluating the modulation of the immune responses against fungi. The evasion of phagocytosis and escape of phagosomes are mechanisms of fungal virulence. Here, we report the methods that can be used for the analysis of the phagocytosis, clearance, and viability of T. stromaticum conidia, a fungus which is used as a biocontrol and biofertilizer agent and is capable of inducing human infections. The protocol consists of 1) Trichoderma culture, 2) washing to obtain conidia, 3) the isolation of peripheral blood mononuclear cells (PBMCs) using the polysucrose solution method and the differentiation of the PBMCs into macrophages, 4) an in vitro phagocytosis method using round glass coverslips and coloration, and 5) a clearance assay to assess the conidia viability after conidia phagocytosis. In summary, these techniques can be used to measure the&#160;fungal clearance efficiency of macrophages.

Author Spotlight: Unraveling the Interplay Between Trichoderma stromaticum and the Mammalian Immune System

The technique involving the phagocytosis of fungal conidia by macrophages is widely used for studies evaluating the modulation of the immune responses against fungi. The purpose of this manuscript is to present a method for evaluating the phagocytosis and clearance abilities of human peripheral blood mononuclear-derived macrophages stimulated with Trichoderma stromaticum conidia.

Macrophages represent a crucial line of defense and are responsible for preventing the growth and colonization of pathogens in different tissues. Conidial ...

Quantitative Flow Cytometric Measurement of Fungal Spore Phagocytosis by Human Phagocytes

Source: Hartung, S., et al., <a href="https://app.jove.com/v/60397">Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry.</a> J. Vis. Exp. (2019)
This video demonstrates a method for quantifying the phagocytosis of FITC-labeled pathogenic fungal spores by human leukocytes. Phagocytosed spores are discerned from cell-adherent spores through counterstaining, followed by flow cytometric analysis.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Phagocytosis Assay

	Incubate 2 x ...

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An Assay for Quantifying Antibody-Mediated Phagocytosis of Parasite-Infected Erythrocytes

Source: Quintana, M. d. P. et al., <a href="https://app.jove.com/v/61538">Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay.</a>&#160;J. Vis. Exp.&#160;(2020)
This video demonstrates a flow cytometry-based phagocytosis assay involving labeled and opsonized Plasmodium falciparum-infected erythrocytes by monocytes. Flow cytometry identifies fluorescence in monocytes, confirming the successful identification of opsonized erythrocytes through phagocytosis.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Parasite culture
NOTE: Follow the ...

Real-Time Assessment of the Antifungal Activity of Primary Human Immune Cells

Source: Brunel, S. F. et al., <a href="https://app.jove.com/v/55444">Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus.</a>J. Vis. Exp. (2017)
This video demonstrates live cell imaging for the analysis of antifungal activity in human neutrophils using fluorescent Aspergillus reporter conidia. Initially, both red and magenta fluorescence are detected within neutrophils, while red fluorescence decreases over time, indicating the degradation of conidia and confirming the antifungal activity of neutrophils.

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

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Chapters in this video

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

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

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung

Viability Assay of Trichoderma stromaticum Conidia Inside Human Peripheral Blood Mononuclear-Derived Macrophages

Quantitative Flow Cytometric Measurement of Fungal Spore Phagocytosis by Human Phagocytes

An Assay for Quantifying Antibody-Mediated Phagocytosis of Parasite-Infected Erythrocytes

Real-Time Assessment of the Antifungal Activity of Primary Human Immune Cells