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All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Purification of human polymorphonuclear leukocytes and isolation of human serum
NOTE: All reagents should be routinely checked for the presence of endotoxin using a commercially available endotoxin detection kit and should contain &#60;25.0 pg/mL endotoxin to prevent unwanted priming of PMNs.
<ol>
	<li>Bring 50 mL of 3% dextran-0.9% NaCl (w/v), 35 mL of 0.9% NaCl (w/v), 20 mL of 1.8% NaCl (w/v), 12 mL of 1.077 g/mL density gradient solution, and 20 mL of injection- or irrigation-grade water to room temperature.</li>
	<li>To isolate human serum, incubate 4 mL of freshly drawn human blood without anti-coagulant at 37 &#176;C in a 15 mL glass tube for 30 min. After incubation, centrifuge the sample at 2,000&#8211;3,000 &#215; g for 10 min at room temperature. Transfer the upper serum layer into a fresh 15 mL conical centrifuge tube and place it on ice.</li>
	<li>Combine 25 mL of freshly drawn heparinized (1000 units/mL) whole human blood with 25 mL of room temperature 3% dextran-0.9% NaCl (1:1 ratio) in two replicate 50 mL conical centrifuge tubes (50 mL total volume per tube). Mix by gently rocking each 50 mL conical tube and then let stand at room temperature for 30 min.</li>
	<li>After incubation at room temperature, two separate layers will appear. Transfer the top layer of each dextran-blood mixture into new 50 mL conical tubes and centrifuge at 450 x g for 10 min at room temperature with low or no brakes.</li>
	<li>Carefully aspirate both supernatants and discard them without disturbing the cell pellets. Gently resuspend each cell pellet in 2 mL of room temperature 0.9% NaCl, combine the resuspended pellets in a single 50 mL conical tube, then add the remaining 0.9% NaCl (final volume of 35 mL).</li>
	<li>Carefully underlay 10 mL of room temperature of 1.077 g/mL density gradient solution beneath the cell suspension using a hand pipette. Spin at 450 x g for 30 min at room temperature with low or no brakes. Gently aspirate the supernatant without disturbing the cell pellet. The supernatant will contain peripheral blood mononuclear cells that can be collected as previously described.</li>
	<li>Lyse the red blood cells by resuspending the cell pellet in 20 mL of room-temperature water. Mix gently by rocking the tube for 30 seconds. The lysis of red blood cells will be accompanied by a distinct decrease in turbidity.</li>
	<li>Immediately add 20 mL of 1.8% NaCl (at room temperature [RT]) and centrifuge sample at 450 &#215; g for 10 min at room temperature. 
	NOTE: It is important to minimize the time that PMNs are left in water alone following red blood cell lysis to maximize PMN yield and prevent PMN lysis and/or activation.</li>
	<li>Carefully aspirate the supernatant without disturbing the cell pellet. Gently resuspend the cell pellet in 2 mL of RT RPMI 1640 medium and place it on ice.</li>
	<li>Count cells using a hemocytometer. Resuspend purified PMNs at a concentration of 1 x 107 cells/mL with ice-cold RPMI and keep them on ice.</li>
	<li>Combine 100 &#181;L of purified PMNs (1 x 106 cells) with 300 &#181;L of ice-cold Dulbecco&#39;s phosphate-buffered saline (DPBS) containing 1 &#181;L of propidium iodide stain in two replicate flow cytometry tubes. For a positive control for plasma membrane damage, add 40 &#181;L of 0.5% Triton X-100 solution into one of the flow cytometry tubes and mix thoroughly.</li>
	<li>Use flow cytometry to measure the forward scatter, side scatter, and propidium iodide staining (excitation/emission maxima at 535/617 nm) of purified cells (Figure 1). 
	NOTE: Forward and side scatter analysis will identify unwanted populations of lymphocytes and monocytes. Propidium iodide will only stain cells with a compromised plasma membrane and purified PMNs that have pronounced populations of propidium iodide-positive cells should not be used. For these studies, purified PMNs were only used if they comprised &#62;98% of purified cells and &#60;5% stained positive for propidium iodide.</li>
	<li>Prepare a 96-well plate for PMN cytotoxicity assays by coating individual wells that will be used in this assay with 100 &#181;L of 20% isolated human serum that has been diluted with DPBS. 
	NOTE: Plating PMNs directly on plastic or glass will cause activation of the cells. Be sure to include at least one negative control well that will only receive media and at least one positive control well that will receive 0.05% Triton X-100.</li>
	<li>Incubate the plate at 37 &#176;C for 30 min. Following incubation, wash the coated wells twice with ice-cold DPBS to remove any excess serum. Gently tap the plate upside down to remove any residual DPBS and place it on ice.</li>
	<li>Gently add 100 &#181;L of purified human PMNs at 1 x 107 cells/mL to each coated well (1 x 106 PMNs/well). Allow PMNs to settle in wells by incubating the plate on ice for at least 5 min. Keep the plate level to allow even distribution of cells in each well and leave it on ice to avoid unwanted activation of PMNs.</li>
</ol>
2. Cytotoxicity assay of S. aureus extracellular proteins against human polymorphonuclear leukocytes
<ol>
	<li>Culture S. aureus overnight in tryptic soy broth (TSB) using a shaking incubator set at 37 &#176;C. For these studies, 20 mL of TSB in separate 150 mL Erlenmeyer flasks were inoculated with frozen cultures of S. aureus strains USA300 or USA300&#916;saeR/S and grown for approximately 14 h with shaking at 250 rpm.</li>
	<li>Subculture S. aureus by performing a 1:100 dilution of overnight bacterial culture with fresh media. Incubate at 37 &#176;C with shaking until the bacteria reach the early stationary growth phase. 
	NOTE: For these experiments, 20 mL of tryptic soy broth in 150 mL Erlenmeyer flasks were inoculated with 200 &#181;L of overnight cultured USA300 or USA300&#916;saeR/S and incubated at 37 &#176;C with shaking at 250 rpm for 5 h.</li>
	<li>When bacteria have reached the early stationary growth phase, transfer 1 mL of subcultured S. aureus into a 1.5 mL microcentrifuge tube and centrifuge at 5,000 &#215; g for 5 min at room temperature.</li>
	<li>Following centrifugation, transfer the supernatant into a 3 mL syringe. Pass supernatants through a 0.22 &#181;m filter and into a new 1.5 mL microcentrifuge tube on ice.</li>
	<li>Perform serial dilutions of supernatants with ice-cold media used to culture S. aureus. 
	NOTE: For the experiments shown, supernatants from USA300 and USA300&#916;saeR/S underwent four consecutive 1/2 log dilutions with ice-cold TSB.</li>
	<li>Gently add supernatant samples or media alone (for negative and positive controls) to individual wells of 96-well plates containing PMNs on ice from step 1.15. For these experiments, 10 &#181;L of USA300 or USA300&#916;saeR/S supernatant samples were added to each well. Gently rock plate to distribute supernatants in wells and incubate at 37 &#176;C.</li>
	<li>At desired times, remove the plate from the incubator and place it on ice. Add 40 &#181;L of 0.5% Triton X-100 to the positive control well.</li>
	<li>Gently pipette the samples up and down in each well to pull off all PMNs adhered to the plate completely, then transfer the samples to flow cytometry tubes on ice that contain 300 &#181;L of ice-cold DPBS with 1 &#181;L of propidium iodide.</li>
	<li>Measure the proportion of propidium iodide-positive PMNs using flow cytometry (Figure 2A). When bound to DNA, propidium iodide has excitation/emission at 535/617 nm.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% Sodium Chloride Injection, USP, 500 mL VIAFLEX Plastic Container</td>
			<td>Baxter</td>
			<td>2B1323Q</td>
			<td>PMN purification</td>
		</tr>
		<tr>
			<td>1.5 mL micro-centrifuge tubes with Snap Caps</td>
			<td>VWR</td>
			<td>89000-044</td>
			<td>Used in washing cells</td>
		</tr>
		<tr>
			<td>1.8% Sodium Chloride Solution</td>
			<td>Sigma-Aldrich</td>
			<td>S5150</td>
			<td>PMN purification</td>
		</tr>
		<tr>
			<td>12x75mm Culture tubes</td>
			<td>VWR</td>
			<td>60818-430</td>
			<td>Used as flow cytometry tubes</td>
		</tr>
		<tr>
			<td>20% (w/w) Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>D8802</td>
			<td>PMN purification</td>
		</tr>
		<tr>
			<td>3125 Hand Tally Counter</td>
			<td>Traceable Products</td>
			<td>3125CC</td>
			<td>For counting cells</td>
		</tr>
		<tr>
			<td>50 mL conical centrifuge tubes</td>
			<td>VWR</td>
			<td>89039-656</td>
			<td>For dispensing media</td>
		</tr>
		<tr>
			<td>Bacto Tryptic Soy Broth, Soybean-Casein Digest Medium</td>
			<td>FischerScientific</td>
			<td>211823</td>
			<td>For growing cell cultures</td>
		</tr>
		<tr>
			<td>BD Disposable Syringes with Luer-Lok Tips, 3 mL</td>
			<td>FischerScientific</td>
			<td>BD 309657</td>
			<td>For filtering supernatants</td>
		</tr>
		<tr>
			<td>Bright-Line Hemocytometer</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629</td>
			<td>Cell counting apparatus</td>
		</tr>
		<tr>
			<td>DPBS, 1x (Dulbecco&#39;s Phosphate Buffered Saline) with calcium and magnesium</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td>Used in washing cells</td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
			<td>PMN purification</td>
		</tr>
		<tr>
			<td>Fisherbrand Sterile Polystyrene Disposable Serological Pipets</td>
			<td>FischerScientific</td>
			<td>13-678-11E</td>
			<td>For aspirating liquid</td>
		</tr>
		<tr>
			<td>Greiner CELLSTAR 96 well plates</td>
			<td>Millipore Sigma</td>
			<td>M0687</td>
			<td>Plate for holding experimental samples</td>
		</tr>
		<tr>
			<td>OMICRON Syringe Filters</td>
			<td>Omicron Scientific</td>
			<td>SFPV13R</td>
			<td>For filtering supernatants</td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>ThermoFisher Scientific</td>
			<td>P3566</td>
			<td>Membrane impermeable DNA stain</td>
		</tr>
		<tr>
			<td>PYREX Brand 4980 Erlenmeyer Flasks</td>
			<td>Cole-Parmer</td>
			<td>EW-34503-24</td>
			<td>For growing cell cultures</td>
		</tr>
		<tr>
			<td>RPMI 1640, 1X without L-glutamine, phenol red</td>
			<td>Corning</td>
			<td>17-105-CV</td>
			<td>Used in resuspending cells</td>
		</tr>
		<tr>
			<td>Sterile Water for Irrigation, USP</td>
			<td>Baxter</td>
			<td>2F7113</td>
			<td>PMN purification</td>
		</tr>
		<tr>
			<td>The Pipette Pump</td>
			<td>Bel-Art Products</td>
			<td>F37898</td>
			<td>For aspirating liquid</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td>Membrane integrity positive control</td>
		</tr>
	</tbody>
</table>

an assay for bacterial extracellular protein-mediated cytotoxicity on pmns

Source: Dankoff, J. G., et al. <a href="https://app.jove.com/v/60681">Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes.</a> J. Vis. Exp. (2020).
This video demonstrates an assay to investigate the cytotoxicity of proteins produced by Staphylococcus aureus, a pathogenic bacterium, on isolated human polymorphonuclear leukocytes (PMNs). The secreted cytotoxic proteins form pores on the PMN cell membrane, initiating cell death. Upon staining the dead cells using propidium iodide, flow cytometry is performed to quantify the dead cells and assess the cytotoxicity of the bacteria.

<img alt="Figure 1" src="/files/ftp_upload/21789/21789_Figure1.jpg" /> 
Figure 1: Flow cytometry analysis of purified PMNs. Representative flow cytometry dot plots of (A) purified human PMNs and (B) PMNs that have been purposely contaminated with peripheral blood mononuclear cells. (C) Representative flow cytometry histogram demonstrating minimal propidium iodide staining (&#60;1%) of purified PMNs (shaded grey) as compared to PMNs treated with 0.05% Triton X-100 (sh...

An Assay for Bacterial Extracellular Protein-Mediated Cytotoxicity on PMNs

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

Take a culture of Staphylococcus aureus, a pathogenic bacteria that secretes leukocidin&#160; &#8212; a pore-forming toxin &#8212; comprising monomeric&#160; S and F components.
Pellet the cells and serially dilute the supernatant. Transfer the leukocidin-containing supernatant into a microplate having polymorphonuclear leukocytes, or PMNs, and incubate.
The S component binds to specific transmembrane receptors on PMNs, followed by hetero-oligomerization with the F component to insert into the cell membrane, resulting in pore formation.
The pore causes a decrease in intracellular potassium ions, activating NLR family pyrin domain-containing 3 or NLRP3 &#160;protein.
NLRP3 binds to apoptosis-associated speck-like protein containing a caspase recruitment domain, or ASC, which recruits pro-caspase-1, forming an inflammasome.
Auto-proteolysis converts pro-caspase-1 to active caspase-1, triggering cell death.
Harvest the cells at defined intervals. Add propidium iodide or PI, which enters dead cells through damaged membranes to bind DNA, emitting fluorescence.
Using flow cytometry, quantify PI-stained cells to assess the cytotoxic effect of both leukocidin concentration and incubation time.

an-assay-for-bacterial-extracellular-protein-mediated-cytotoxicity-on

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

52 ビュー. 出典:Dankoff, J. G., et al. ヒト多形核白血球に対する黄色ブドウ球菌の細胞毒性の定量化。 J. Vis. Exp.(2020年)。 このビデオは、病原性細菌である黄色ブドウ球菌が単離されたヒト多形核白血球(PMN)で産生するタンパク質の細胞毒性を調査するアッセイを示しています。分泌された細胞傷害性タンパク質は、PMN細胞膜上に細孔を形成し、細胞死を開始します。ヨウ化プロピジウム...

ビデオ: PMNに対する細菌の細胞外タンパク質媒介性細胞毒性のアッセイ - 概念

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

感染した組織の細菌の遺伝子の規則を勉強する蛍光ベースのメソッド

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

JoVE Journal

遺伝学

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during infection.

哺乳動物宿主細胞に分泌された細菌毒素の影響を研究するために、透過膜を挿入ベースの感染システムの実現

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

免疫・感染学

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of infection where they use processes such as phagocytosis, production of reactive oxygen and nitrogen species (ROS, RNS, respectively), NETosis (neutrophil extracellular trap), and degranulation to kill microbes and resolve the infection. Interactions between neutrophils and planktonic microbes have been extensively studied. There have been emerging interests in studying infections caused by biofilms in recent years. Biofilms exhibit properties, including tolerance to killing by neutrophils, distinct from their planktonic-grown counterparts. With the successful establishment of both in vitro and in vivo biofilm models, interactions between these microbial communities with different immune cells can now be investigated. Here, techniques that use a combination of traditional biofilm models and well-established neutrophil activity assays are tailored specifically to study neutrophil and biofilm interactions. Wide-field fluorescence microscopy is used to monitor the localization of neutrophils in biofilms. These biofilms are grown in static conditions, followed by the addition of neutrophils derived from human peripheral blood. The samples are stained with appropriate dyes prior to visualization under the microscope. Additionally, the production of ROS, which is one of the many neutrophil responses against pathogens, is quantified in the presence of a biofilm. The addition of immune cells to this established system will expand the understanding of host-pathogen interactions while ensuring the use of standardized and optimized conditions to measure these processes accurately.

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

The present protocol describes the study of neutrophil-biofilm interactions. Staphylococcus aureus biofilms are established in vitro and incubated with peripheral blood-derived human neutrophils. The oxidative burst response from neutrophils is quantified, and the neutrophil localization within the biofilm is determined by microscopy.

ヒト好中球と黄色ブドウ球菌バイオフィルムの間の相互作用を視覚化および定量するための標準化されたin vitroアッセイ

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of ...

An Assay for Bacterial Extracellular Protein-Mediated Cytotoxicity on PMNs

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