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>

<ol>
	<li>Brown, G. 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=Hidden+killers:+human+fungal+infections.">Hidden killers: human fungal infections.</a> Science Translational Medicine. 4 (165), 113 (2012).</li><li>Brakhage, A. A., Bruns, S., Thywissen, A., Zipfel, P. F., Behnsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+phagocytes+with+filamentous+fungi.">Interaction of phagocytes with filamentous fungi.</a> Current Opinion in Microbiology. 13 (4), 409-415 (2010).</li><li>Heinekamp, T., 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=Interference+of+Aspergillus+fumigatus+with+the+immune+response.">Interference of Aspergillus fumigatus with the immune response.</a> Seminars in Immunopathology. 37 (2), 141-152 (2015).</li><li>Slesiona, 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=Persistence+versus+escape:+Aspergillus+terreus+and+Aspergillus+fumigatus+employ+different+strategies+during+interactions+with+macrophages.">Persistence versus escape: Aspergillus terreus and Aspergillus fumigatus employ different strategies during interactions with macrophages.</a> PLoS One. 7 (2), 31223 (2012).</li><li>Busetto, S., Trevisan, E., Patriarca, P., Menegazzi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single-step,+sensitive+flow+cytofluorometric+assay+for+the+simultaneous+assessment+of+membrane-bound+and+ingested+Candida+albicans+in+phagocytosing+neutrophils.">A single-step, sensitive flow cytofluorometric assay for the simultaneous assessment of membrane-bound and ingested Candida albicans in phagocytosing neutrophils.</a> Cytometry A. 58 (2), 201-206 (2004).</li><li>Lowe, D. 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. G., D'Amelio, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New,+simple+flow+cytometry+technique+to+discriminate+between+internalized+and+membrane-bound+particles+in+phagocytosis.">New, simple flow cytometry technique to discriminate between internalized and membrane-bound particles in phagocytosis.</a> Cytometry. 10 (3), 320-325 (1989).</li><li>Heinzelmann, M., Gardner, S. A., Mercer-Jones, M., Roll, A. J., Polk, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+phagocytosis+in+human+neutrophils+by+flow+cytometry.">Quantification of phagocytosis in human neutrophils by flow cytometry.</a> Microbiology and Immunology. 43 (6), 505-512 (1999).</li><li>Perticarari, S., Presani, G., Mangiarotti, M. A., Banfi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+flow+cytometric+method+to+measure+phagocytosis+and+oxidative+products+by+neutrophils.">Simultaneous flow cytometric method to measure phagocytosis and oxidative products by neutrophils.</a> Cytometry. 12 (7), 687-693 (1991).</li><li>Sveum, R. J., Chused, T. M., Frank, M. M., Brown, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+fluorescent+method+for+measurement+of+bacterial+adherence+and+phagocytosis.">A quantitative fluorescent method for measurement of bacterial adherence and phagocytosis.</a> Journal of Immunolological Methods. 90 (2), 257-264 (1986).</li><li>de Boer, E. C., Bevers, R. F., Kurth, K. H., Schamhart, D. 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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 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 ...

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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 ...

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Research

JoVE Journal

Immunology and Infection

6.6K 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

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 &#176;C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.

We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots ("H" and "F") of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.

The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

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

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

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

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

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Measuring Erythrocyte Complement Receptor 1 Using Flow Cytometry

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, transports immune complexes, and participates in humoral and cellular immune responses. CR1 is present on the surface of many cell types, including erythrocytes, and exhibits polymorphisms in length, structure (Knops, or KN, blood group), and density. The average density of CR1 per erythrocyte (CR1/E) is 500 molecules per erythrocyte. This density varies from one individual to another (100&#8211;1,200 CR1/E) and from one erythrocyte to another in the same individual. We present here a robust flow cytometry method to measure the density of CR1/E, including in subjects expressing a low density, with the help of an amplifying immunostaining system. This method has enabled us to show the lowering of CR1 erythrocyte expression in diseases such as Alzheimer&#39;s disease (AD), systemic lupus erythematosus (SLE), AIDS, or malaria.

The aim of this method is to determine the CR1 density in the erythrocytes of any subject by comparing with three subjects whose erythrocyte CR1 density is known. The method uses flow cytometry after immunostaining of the subjects&#39; erythrocytes by an anti-CR1 monoclonal antibody coupled to an amplified system using phycoerythrin (PE).

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, ...

Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is responsible for altering infection for many of these viruses. Thus, characterizing the nature of virus-bacteria binding is a foundational step needed for elucidating the mechanism(s) by which bacteria alter viral infection. For human norovirus, commensal bacteria enhance B cell infection. The virus directly binds to these bacteria, indicating that this direct interaction is involved in the mechanism of infection enhancement. A variety of techniques can be used to quantify interactions between bacteria and viruses including scintillation counting of radiolabeled viruses and polymerase chain reaction (PCR). Both methods require the use of live virus, which may need to be generated in the laboratory. Currently, none of the established in vitro culture systems available for human norovirus are robust enough to allow for generation of highly concentrated viral stocks. In lieu of live virus, virus-like particles (VLPs) have been used to characterize the interactions between norovirus and bacteria. Herein a flow cytometry method is described with uses virus specific antibodies to quantify VLP binding to gram-negative and gram-positive bacteria. Inclusion of both bacteria only and isotype controls allowed for optimization of the assay to reduce background antibody binding and accurate quantification of VLP attachment to the bacteria tested. High VLP:bacterium ratios result in VLPs binding to large percentages of the bacterial population. However, when VLP quantities are decreased, the percent of bacteria bound also decreases. Ultimately, this method can be employed in future experiments elucidating the specific conditions and structural components that regulate norovirus:bacterial interactions.

The goal of this protocol is to quantify binding of the eukaryotic pathogen human norovirus to bacteria. After performing an initial virus-bacterium attachment assay, flow cytometry is used to detect virally-bound bacteria within the population.

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is ...

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 ...