Name: measuring phagocytosis of aspergillus fumigatus conidia by human leukocytes using flow cytometry
Uploaded: 2019-12-07T12:00:00
Description: Watch this Scientific Journal Video about Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry at JoVE.com

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. fu...

<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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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+fluorescent+flow+cytometric+assessment+of+bacterial+internalization+and+binding+by+epithelial+cells.">Double fluorescent flow cytometric assessment of bacterial internalization and binding by epithelial cells.</a> Cytometry. 25 (4), 381-387 (1996).</li><li>Hartung, 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=Fast+and+Quantitative+Evaluation+of+Human+Leukocyte+Interaction+with+Aspergillus+fumigatus+Conidia+by+Flow+Cytometry.">Fast and Quantitative Evaluation of Human Leukocyte Interaction with Aspergillus fumigatus Conidia by Flow Cytometry.</a> Cytometry A. 95 (3), 332-338 (2019).</li><li>Fei, C., Lillico, D. M. E., Hall, B., Rieger, A. M., Stafford, J. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#181;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 &#176;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 ...

This protocol is significant because it quantitatively measures phagocytosis of labeled Aspergillus fumigateurs Conidia by human leukocytes, along with the attachment of Conidia to cells, and lack of interaction by flow cytometry. The main advantage of this method is that it facilitates the fast analysis of a large number of cells. In addition, labeling of the cell markers allows to separate assessment of neutrophils and monocytes within the same sample.In any this technique can be used to analyze other clinical relevant fungi like Mucorales. Instead red blood cells might be used as phagocytes. To begin this procedure, wet a paper towel with disinfectant, and place it into a bio safety cabinet.Place a plate of grown Aspergillus fumigatus on top of the paper towel to prevent over-distribution of volatile Conidia. Add 10 milliliters of PBS containing 0.01%detergent on top of the fungus, and use a Drigalski spatula to spread the liquid over the plate, and rub off the dark colored Conidia. Transfer the Conidia suspension to a 50 milliliter through a 30 micrometer strainer, which will remove any residual mycelia.Add another 10 milliliters of PBS with detergent to the plate, spread it with a spatula, and transfer it to the same tube through the strainer. Next, prepare a sterile solution of 0.1 molar sodium carbonate, dissolved in PBS. And prepare a 0.1 millimolar solution of FITC powder, in this sodium carbonate solution.Re-suspend 100 million or less Conidia, in five milliliters of this FITC solution, in a 15 milliliters tube. Incubate in a rotator at 37 degrees Celsius for 20 minutes. To swell to Conidia, resuspend the FITC labeled Conidia in five milliliters of RPMI medium, supplemented with 10%FCS.And incubate in a rotator at 37 degrees Celsius for the desired time. In a bio safety cabinet, place five milliliters of buffy coat into a 50 milliliters tube. Fill up the tube with EL buffer, and invert three times.Incubate horizontally for five to eight minutes, until the milky appearance of the mixture turns clear. Then, centrifuge at 300 times G, and at room temperature for 10 minutes. Discard the supernatant and resuspend the pellet in one milliliters of EL buffer by pipetting.Add another 24 milliliters of EL buffer, and invert the tube several times. Centrifuge at 300 times G and at room temperature for five minutes. Discard the supernatant and resuspend the cells in one milliliters of RPMI media, supplemented with 10%FCS.To begin, transfer two million leukocytes and four million FITC-labeled Conidia in 1.5 milliliters of RPMI supplemented with 10%FCS, to a 12-well culture plate. Include a control of cells only with no Conidia, and a control of cells with unlabeled Conidia. Incubate the plate in a humidified carbon dioxide incubator, at 37 degrees Celsius for the desired amount of time.When the incubation is complete, use a cell scraper to harvest the cells, and transfer them to a 15 milliliters tube. First, put 100 microliters of each sample and controls into one well of a 96-well V-bottom plate. Add 150 microliters of PBS containing two millimolar of EDTA for washing.For color compensation, place one million cells without Conidia for each color, in other wells of the plate. Include a well of cells that will be left unstrained. Add 150 microliters of PBS containing two millimolar of EDTA to each well for washing.Next, cover the plate with an adhesive foil, and centrifuge at 300 times G and at room temperature for five minutes. Then, remove the foil and discard the supernatant by quickly and forcefully inverting the plate only once over either the sink, or a disposable paper towel. Resuspend the cells in 100 microliters of antibody mix, and mix well by pipetting.For color compensations, resuspend the respective cells in 100 microliters of PBS containing two millimolar of EDTA, and add a single antibody type to each well, at the same amount used in the antibody mix. Cover with an adhesive foil, and incubate at room temperature in the dark for 20 minutes. After this, remove the foil and add 150 microliters of PBS containing two millimolar of EDTA to each well for washing.Cover the plate with adhesive foil again. Centrifuge at 300 times G and at room temperature for five minutes. Then remove the foil, and discard the supernatant by quickly and forcefully inverting the plate over either a sink or disposable paper towel.Resuspend the cells in 200 microliters of PBS containing 2 millimolar EDTA, and transfer the cell suspension from each well, into a separate round bottom tube. Start the flow cytometer, and let it warm up. In the acquisition software, create a new experiment, and set up and label the new samples.Set up the parameters and the detectors for the appropriate fluorophores, as outlined in the text protocol. In this software, display the appropriate dot plots as outlined in the text protocol. Using the cells only sample, acquire some cells and set the gate around leukocytes.Based on the leukocyte gate, gate for CD45 positive cells, to separate from Conidia in the SSC CD45 plot. In a dot plot, CD14, CD66b, gate neutrophils and monocytes separately. After this, change from the cells only sample, to unlabeled Conidia.In a dot plot, anti-FITC, APC FITC, display neutrophils in set quadrants for anti-FITC, and FITC signals. Open the statistics view and adjust the quadrants, allowing a maximum of 1%of cells in the quadrants Q1, Q2, and Q4.Repeat this process for the monocyte gate. In the leukocyte gate, record all samples with at least 20, 000 events.Phagocytosis of FITC-labeled Conidia by human neutrophils can be read from the statistics views. In this study, a fast flow cytometric method is used to measure the interaction of Aspergillus fumigateurs Conidia with a large number of primary human leukocytes. Using the appropriate antibodies and the gating strategy shown here, a general gating of human leukocytes by FSC and SSC characteristics, is followed by a separation of leukocytes in free Conidia, by the pan leukocyte marker, CD45.Conidia can reach almost cell size at the time of flow cytometry, especially when using swollen Conidia, and or long incubation times, and thus can buy us analysis. Since human primary monocytes and neutrophils take up Conidia differently, this protocol allows for the separate analysis of these cell population based, on staining with the well-established cell lineage markers, CD14 for monocytes, and CD66b for neutrophils. The percentage of human primary phagocytes internalizing Conidia can be highly variable among blood donors, but also depends on experimental factors, such as incubation time and swelling state of Conidia.Spore internalization can be detected after 0.5 hours of co-incubation, and increases with time. Pre-swollen Conidia are taken up easier than resting spores, even at short incubation times. If Conidia are fixed with formaldehyde, phagocytosis is diminished compared to native Conidia.Following this procedure, supernatants of co-incubated immune cells and Conidia can be collected and analyzed by Eliza to check if the interaction elicits a cytokine release by the immune cells. Please remember, human blood can potentially transmit infectious diseases. This work requires vaccination across Hepatitis B, the use of a bio safety cabinet, as well as wearing gloves at a lab coat.

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