quantitative flow cytometric measurement of fungal spore phagocytosis by human phagocytes

Quantitative Flow Cytometric Measurement of Fungal Spore Phagocytosis by Human Phagocytes

To begin, transfer 2 million leukocytes and 4 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 centigrade 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-milliliter tube.
First, put 100 microliters of each sample and control into one well of a 96-well V-bottom plate. Add 150 microliters of PBS containing 2 millimolar of EDTA for washing. For color compensation, place 1 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 2 millimolar of EDTA to each well for washing.
Next, cover the plate with an adhesive foil and centrifuge at 300 times g 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 compensation, resuspend the respective cells in 100 microliters of PBS containing 2 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 2 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 a 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 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/FITC display neutrophils, and 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.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. 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>
2. 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 the 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 with 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>
3. Flow Cytometry
<ol>
	<li>Start the flow cytometer and let it warm up. Start the acquisition software.</li>
	<li>Create a new experiment and set up and label the samples.</li>
	<li>Set up parameters (FSC 250, SSC 250) and detectors for fluorophores FITC, APC, BUV395, V500, and 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 it 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 a 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 3.5.4 and 3.5.5 for the monocyte gate.</li>
		<li>Record all samples with at least 20,000 events in the leukocyte gate.</li>
	</ol>
	</li>
</ol>
Table 1: Antibody mix for flow cytometry. Amounts of each reagent are given as microliters per sample (1 x 106 cells) to be analyzed.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>&#181;l per sample</td>
		</tr>
		<tr>
			<td>CD45 BUV395</td>
			<td>1</td>
		</tr>
		<tr>
			<td>CD14 V500</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>CD66b PerCP-Cy5.5</td>
			<td>1.5</td>
		</tr>
		<tr>
			<td>anti-FITC APC</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>PBS + 2 mM EDTA</td>
			<td>97</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>100</td>
		</tr>
	</tbody>
</table>

<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>Cell culture plate, 12-well</td>
			<td>Greiner Bio-one</td>
			<td>665180</td>
			<td></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>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma Aldrich</td>
			<td>ED3SS-500g</td>
			<td>2 mM in PBS</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>Phosphate 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>Software for data acquisition and analysis</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>V-bottom plate, 96 well</td>
			<td>Brand</td>
			<td>781601</td>
			<td>Untreated surface</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Carl Roth</td>
			<td>PO87.3</td>
			<td>Histofix</td>
		</tr>
	</tbody>
</table>

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 ...

Take a multi-well plate containing a human leukocyte suspension. Add FITC-labeled pathogenic fungal spores.
During incubation, the spores swell and break their rigid hydrophobic protein layer, exposing the cell wall polysaccharides recognized by specific pattern recognition receptors on phagocytic cells.
This binding triggers intracellular signaling pathways, leading to actin rearrangement and spore internalization within phagosomes.
Depending on the activation state of phagocytes, some spores remain adhered to the cells without internalization.
Post-incubation, harvest the cells and transfer them to a fresh tube.
Centrifuge. Resuspend the cells in a buffer and transfer them to a multi-well V-bottom plate.
Introduce fluorophore-labeled anti-FITC antibodies that bind to the exposed FITC molecules on adhering spores, counterstaining them.
Centrifuge. Resuspend the cells in a buffer and transfer them to flow cytometer tubes.
Perform flow cytometry to differentiate between internalized and cell-adherent spores.
Phagocytes containing internalized spores display FITC signals, while cells with adherent spores exhibit FITC and counterstain signals.

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视频: 人吞噬细胞真菌孢子吞噬作用的定量流式细胞术测量 - 实验

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.

为了应对抑菌活性的实时成像人力初级中性粒细胞和单核细胞<em&gt;一种。曲霉</em

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

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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.

实验性曲霉病肺真菌负担的组织学定量测定

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

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major immune cells and only phagocytes in the brain, recent studies have shown that astrocytes also participate in various phagocytic processes, such as developmental synapse elimination and clearance of amyloid beta plaques in Alzheimer&#39;s disease (AD). Despite these findings, the efficiency of astrocyte engulfment and degradation of their targets is unclear compared with that of microglia. This lack of information is mostly due to the lack of an assay system in which the kinetics of astrocyte- and microglia-mediated phagocytosis are easily comparable. To achieve this goal, we have developed a long-term live-imaging in vitro phagocytosis assay to evaluate the phagocytic capacity of purified astrocytes and microglia. In this assay, real-time detection of engulfment and degradation is possible using pH indicator-conjugated synaptosomes, which emit bright red fluorescence in acidic organelles, such as lysosomes. Our novel assay provides simple and effective detection of phagocytosis through live-imaging. In addition, this in vitro phagocytosis assay can be used as a screening platform to identify chemicals and compounds that can enhance or inhibit the phagocytic capacity of astrocytes. As synaptic pruning malfunction and pathogenic protein accumulation have been shown to cause mental disorders or neurodegenerative diseases, chemicals and compounds that modulate the phagocytic capacity of glial cells should be helpful in treating various neurological disorders.

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

This protocol presents an in vitro live-imaging phagocytosis assay to measure the phagocytic capacity of astrocytes. Purified rat astrocytes and microglia are used along with pH indicator-conjugated synaptosomes. This method can detect real-time engulfment and degradation kinetics and provides a suitable screening platform to identify factors modulating astrocyte phagocytosis.

一种新的体外活体成像法测定星形胶质细胞的吞噬作用 pH 指标-共轭突

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major ...

Quantitative Flow Cytometric Measurement of Fungal Spore Phagocytosis by Human Phagocytes

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Quantitative Flow Cytometric Measurement of Fungal Spore Phagocytosis by Human Phagocytes