This work was supported by a Leading Initiative for Excellent Young Researchers (LEADER, No. 16812285) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, a Grant-in-Aid for Young Scientist Research (No. 18K18157, 16K21034) from Japan Society for the Promotion of Science (JSPS) to M.M., and Grant-in-Aid from MEXT to K.K. (No. 17H06417, 17H06413).

1. Preparation of GVs Containing Bacterial Cells by the Droplet Transfer Method
<ol>
	<li>Prepare lipid stock solutions of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, 10 mM, 1 mL) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000] (biotin-PEG-DSPE, 0.1 mM, 1 mL) in chloroform/methanol solution (2/1, v/v) and store the stock at -20 &#176;C.</li>
	<li>Preparation of a lipid-containing oil solution
	<ol>
		<li>Pour 20 &#956;L of the POPC solution and 4 &#956;L of the biotin-PEG-DSPE solution into a glass tube (Figure 1b (i)).</li>
		<li>Evaporate the organic solvent by air flow to form a lipid film and place the film in a desiccator for 1 h to completely evaporate the organic solvent (Figure 1b (ii)). 
		NOTE: It is necessary to evaporate the organic solvent in a fume hood.</li>
		<li>Add 200 &#956;L of mineral oil (0.84 g/mL, Table of Materials) to the glass vial (Figure 1b (iii)).</li>
		<li>Wrap the opening part of the glass vial with film and sonicate it in an ultrasonic bath (120 W) for at least 1 h (Figure 1b (iii)). The final concentrations of POPC and biotin-PEG-DSPE are 1 mM and 0.002 mM, respectively.</li>
	</ol>
	</li>
	<li>Pre-culture of bacterial cells
	<ol>
		<li>Inoculate E. coli into 1x LB medium (1 g yeast extract, 2 g bacto tryptone, and 2 g sodium chloride in 200 mL of deionized water) from an LB plate and incubate at 37 &#176;C for 12&#8211;14 h (overnight).</li>
		<li>After incubation, collect 20 &#956;L of the culture solution and transfer to 1.98 mL of fresh 1x LB medium, and culture the cells again for 2 h.</li>
		<li>Check the optical density at 600 nm (OD600) value of the pre-culture solution (prepared in step 1.3.2). A pre-culture solution of OD600 = 1.0&#8211;1.5 should be used.</li>
	</ol>
	</li>
	<li>Preparation of the outer and inner aqueous solutions of GVs
	<ol>
		<li>Dissolve glucose in 1x LB medium to prepare an outer aqueous solution of GVs. Prepare 20 mL of a stock glucose solution (500 mM).</li>
		<li>Dilute the stock glucose solution with 1x LB medium to 200 mM (Table 1).</li>
		<li>Dissolve sucrose in 1x LB medium to prepare an inner aqueous solution of GVs. Prepare 20 mL of a stock sucrose solution (500 mM).</li>
		<li>Mix the pre-culture solution (OD600 = 1.0&#8211;1.5), sucrose solution (500 mM), and 1x LB medium (Table 1). The final OD600 value of the culture solution should be 0.01&#8211;0.015 and the final sucrose concentration should be 200 mM. 
		NOTE: Take care to avoid osmotic pressure. It is necessary to balance the concentration between the inner and outer aqueous solution.</li>
	</ol>
	</li>
	<li>Preparation of water-in-oil (W/O) droplets containing bacterial cells
	<ol>
		<li>Add 2 &#956;L of the inner aqueous solution of GVs (prepared in step 1.4.4) to 50 &#956;L of the oil solution containing lipids (mineral oil with POPC and biotin-PEG-DSPE) in a 0.6 mL lidded plastic tube (Figure 1b (iv)).</li>
		<li>Emulsify the two components in the plastic tube by tapping the tube by hand (Figure 1b (v)).</li>
	</ol>
	</li>
	<li>Formation of GVs containing bacterial cells
	<ol>
		<li>Add 50 &#956;L of the outer aqueous solution of GVs (prepared in step 1.4.2) in a 1.5 mL lidded plastic tube (Figure 1b (vi)) and gently layer 150 &#956;L of the oil solution containing lipids (mineral oil with POPC and biotin-PEG-DSPE) on the surface of the outer aqueous solution (Figure 1b (vii)). Incubate this sample at room temperature (RT, 25 &#176;C) for 10&#8211;15 min. Check to ensure that the interface of the oil and aqueous solutions is flat.</li>
		<li>Add 50 &#956;L of the W/O droplet solution (prepared in step 1.5.2) on the interface of the oil and aqueous solution using a pipette (Figure 1b (viii)).</li>
		<li>Centrifuge the 1.5 mL lidded plastic tube (from step 1.6.2) for 10 min at 1,600 x g at RT in a desktop centrifuge (Figure 1b (ix)). After centrifugation, aspirate the oil (top layer) from the 1.5-mL lidded plastic tube using a pipette, and collect the GVs containing bacterial cells (Figure 1b (x)).</li>
	</ol>
	</li>
</ol>
2. Preparation of a GV Observation System (Bacterial Cell Culture System)
<ol>
	<li>Preparation of small vesicles (SVs) for constructing a supported bilayer membrane
	<ol>
		<li>Pour 20 &#956;L of the POPC solution and 4 &#956;L of the biotin-PEG-DSPE solution into a glass tube (using the same lipid composition as used for GV preparation in step 1.1).</li>
		<li>Evaporate the organic solvent by air flow to form a lipid film and place this sample in a desiccator for 1 h to completely evaporate the organic solvent.</li>
		<li>Add 200 &#956;L of 200 mM glucose in 1x LB medium (the outer aqueous solution of GVs) to the glass vial.</li>
		<li>Wrap the opening part of the glass vial with film and sonicate it in an ultrasonic bath (120 W) for at least 1 h.</li>
		<li>Prepare SVs by the extrusion method21 using a mini-extruder and polycarbonate membrane with 100 nm pore size.</li>
	</ol>
	</li>
	<li>Preparation of a handmade chamber
	<ol>
		<li>Drill a 7 mm hole with a hollow punch on a double-faced seal (10 mm x 10 mm x 1 mm).</li>
		<li>Paste the double-faced seal with the hole on a cover glass (30 mm x 40 mm, thickness 0.25&#8211;0.35 mm).</li>
	</ol>
	</li>
	<li>Preparation of a supported bilayer membrane on the cover glass in the hole of the chamber
	<ol>
		<li>Add 30 &#956;L of the SV solution to the hole of the chamber (prepared in section 2.2) and incubate at RT for 30 min.</li>
		<li>Gently wash the hole twice with 20 &#956;L of 1x LB medium containing 200 mM glucose (the outer aqueous solution of GVs) by pipetting.</li>
	</ol>
	</li>
	<li>Immobilization of GVs on the supported bilayer membrane on the cover glass in the hole of the chamber
	<ol>
		<li>Introduce 10 &#956;L of neutravidin with the outer aqueous solution of GVs (1 mg/mL) into the hole and incubate at RT for 15 min.</li>
		<li>Gently wash the hole twice with 20 &#956;L of 1x LB medium containing 200 mM glucose (the outer aqueous solution of GVs) by pipetting.</li>
		<li>Add all solution containing GVs (prepared in step 1.6.3) into the hole of the chamber and seal with a cover glass (18 mm x 18 mm, thickness 0.13&#8211;0.17 mm) (Figure 2b).</li>
	</ol>
	</li>
	<li>Microscopic observation of bacterial cell growth inside GVs
	<ol>
		<li>Set a microscopic heating stage system with an inverted microscope equipped with a 40x/0.6 numerical aperture (NA) objective lens with a long working distance (Figure 2b).</li>
		<li>Place the chamber on the microscopic heating stage system (Figure 2b). Incubate the GVs containing bacterial cells in the chamber at a static condition for 6 h at 37 &#176;C.</li>
		<li>Capture and record microscope images of bacterial cell growth inside GVs every 30 min by using a scientific complementary metal oxide semiconductor (sCMOS) camera.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Here, we describe a method for culturing bacterial cells at the single-cell level inside GVs. This simple method involves forming GVs containing bacterial cells at the single-cell level by using the droplet transfer method. Compared with other approaches for obtaining GVs containing bacterial cells, this method has two advantages: (i) it is easy to develop, and (ii) a small volume (2 &#956;L) of the sample solution is required to prepare the GVs. The droplet transfer method20 for preparing GVs con...

The culture of bacterial cells at the single-cell level has received increasing attention. Culturing bacterial cells at the single-cell level inside a confined space can elucidate bacterial functions such as phenotypic variability1,2,3,4, cell behavior5,6,7,8,9, and antibiotic resistance10,11. Because of recent advances in culture techniques, the culture of single bacteria can be achieved inside a confined space, such as in a well-chip4,7,8, gel droplet12,13, and water-in-oil (W/O) droplet5,11. To promote understanding or utilization of single bacterial cells, further technical developments of cultivation techniques are needed.
Vesicles that mimic the biological cell membrane are spherical compartments consisting of amphiphilic molecules and can hold various materials. Vesicles are classified according to size and include small vesicles (SVs, diameter &#60; 100 nm), large vesicles (LVs, &#60;1 &#956;m), and giant vesicles (GVs, &#62;1 &#956;m). SVs or LVs are commonly used as drug carriers because of their affinity to the biological cell membrane14. GVs have also been used as a reactor system for the construction of protocells15 or artificial-cells16. Encapsulation of biological cells into GVs has been reported17,18, and thus GVs show potential as a cell culture system when combined with the reactor system.
Here, along with a video of experimental procedures, we describe how GVs can be used as novel cell-culture vessels19. GVs containing bacteria were made by the droplet transfer method20 and were then immobilized on a supported membrane on a cover glass. We used this system to observe bacterial growth at the single-cell level inside GVs in real-time.

<table>
	<tbody>
		<tr>
			<td>Bactotryptone</td>
			<td>BD Biosciences</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Wako Pure Chemicals</td>
			<td>032-21921</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass (18 &#215; 18 mm)</td>
			<td>Matsunami Glass Ind.</td>
			<td>C018181</td>
			<td>thickness 0.13&#8211;0.17 mm</td>
		</tr>
		<tr>
			<td>Cover glass (30 &#215; 40 mm)</td>
			<td>Matsunami Glass Ind.</td>
			<td>custom-order</td>
			<td>thickness 0.25&#8211;0.35 mm</td>
		</tr>
		<tr>
			<td>Desktop centrifuge</td>
			<td>Hi-Tech Co.</td>
			<td>ATT101</td>
			<td>swing rotor type</td>
		</tr>
		<tr>
			<td>Double-faced seal (10 &#215; 10 &#215; 1 mm)</td>
			<td>Nitoms</td>
			<td>T4613</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vial</td>
			<td>AS ONE</td>
			<td>6-306-01</td>
			<td>Durham fermentation tube</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Wako Pure Chemicals</td>
			<td>049-31165</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Olympus</td>
			<td>IX-73</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Wako Pure Chemicals</td>
			<td>133-16771</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopic heating stage system</td>
			<td>TOKAI HIT</td>
			<td>TP-110R-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Nacalai Tesque</td>
			<td>23334-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-extruder</td>
			<td>Avanti Polar Lipids</td>
			<td>610000</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutravidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>31000</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective lens</td>
			<td>Olympus</td>
			<td>LUCPLFLN 40&#215;/0.6 NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate membranes</td>
			<td>Avanti Polar Lipids</td>
			<td>610005</td>
			<td>pore size 100 nm</td>
		</tr>
		<tr>
			<td>sCMOS camera</td>
			<td>Andor</td>
			<td>Zyla 4.2 plus</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Wako Pure Chemicals</td>
			<td>191-01665</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Wako Pure Chemicals</td>
			<td>196-00015</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic bath</td>
			<td>AS ONE</td>
			<td>ASU-3D</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>BD Biosciences</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>0.6 mL lidded plastic tube</td>
			<td>Watson</td>
			<td>130-806C</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL lidded plastic tube</td>
			<td>Sumitomo Bakelite Co.</td>
			<td>MS4265-M</td>
			<td></td>
		</tr>
		<tr>
			<td>1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline</td>
			<td>Avanti Polar Lipids</td>
			<td>850457P</td>
			<td>POPC</td>
		</tr>
		<tr>
			<td>1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000]</td>
			<td>Avanti Polar Lipids</td>
			<td>880129P</td>
			<td>Biotin-PEG-DSPE</td>
		</tr>
	</tbody>
</table>

bacterial cell culture at the single-cell level inside giant vesicles

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for understanding the function of bacterial cells in the natural environment. Because of technological advances, various bacterial cell functions can be revealed at the single-cell level inside a confined space. GVs are spherical micro-sized compartments composed of amphiphilic lipid molecules and can hold various materials, including cells. In this study, a single bacterial cell was encapsulated into 10&#8211;30 &#956;m GVs by the droplet transfer method and the GVs containing bacterial cells were immobilized on a supported membrane on a glass substrate. Our method is useful for observing the real-time growth of single bacteria inside GVs. We cultured Escherichia coli (E. coli) cells as a model inside GVs, but this method can be adapted to other cell types. Our method can be used in the science and industrial fields of microbiology, biology, biotechnology, and synthetic biology.

We present a simple method for generating GVs containing single bacterial cells using the droplet transfer method (Figure 1). Figure 1a shows a schematic image of the precipitation of GVs containing bacteria. W/O droplets containing bacteria are transferred across the oil-water (lipid monolayer) interface by centrifugation to form GVs. The difference in density between sucrose (inner aqueous solution) and glucose (outer aqueous s...

Watch this Scientific Journal Video about Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles at JoVE.com

Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles

Cultivo celular bacteriano en el nivel de una sola célula dentro de las vesículas gigantes

We demonstrate single-cell culture of bacteria inside giant vesicles (GVs). GVs containing bacterial cells were prepared by the droplet transfer method and were immobilized on a supported membrane on a glass substrate for direct observation of bacterial growth. This approach may also be adaptable to other cells.

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for ...

bacterial-cell-culture-at-the-single-cell-level-inside-giant-vesicles

Research

JoVE Journal

Immunology and Infection

6.8K vistas.  National Institute of Advanced Industrial Science and Technology (AIST). Este protocolo proporciona un medio para el cultivo de células bacterianas a nivel de una sola célula dentro de las vesículas gigantes. Es fácil desarrollar las vesículas gigantes, y sólo se requiere un volumen muy pequeño de solución de muestra para preparar las vesículas. Demostrando el procedimiento estará Yuri Ota, un candidato al doctorado de nuestro laboratorio.Comience añadiendo 20 microlitros de solución POPC recién preparada y cuatro microlitros de biotina-PEG-DSP...

Video: Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Evaluación De La Invasión Bacteriana De Las Células Cardíacas En Cultivo Y Colonización Del Corazón En Ratones Infectados Usando Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Investigación

Inmunología e Infección

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Generación y multi-fenotípicos cribado de alto contenido de<em&gt; Coxiella burnetii</em&gt; transposón mutantes

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

El desarrollo y la identificación de una subpoblación de Novela derivada de neutrófilos humanos fagocitos gigante<em&gt; In Vitro</em

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite&#39;s ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/&#916;Lmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

Cuantificación del crecimiento intracelular dentro de los macrófagos es un rápido y confiable método para evaluar la virulencia de los parásitos de Leishmania

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Cuantificación de Efferocytosis por microscopía de fluorescencia unicelular

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

Usando un patógeno bacteriano a punta de prueba para las respuestas celular y organísmica nivel Host

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

Aislamiento de las comunidades bacterianas intracelulares solo generados a partir de un modelo murino de infección del tracto urinario para posteriores análisis unicelular

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Un alto rendimiento Shigella-ensayo bactericida específico

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Electrophysiological Recordings of Single-cell Ion Currents Under Well-defined Shear Stress

Fluid shear stress is well known to play a major role in endothelial function. In most vascular beds, elevated shear stress from acute increases in blood flow triggers a signaling cascade resulting in vasodilation thereby alleviating mechanical stress on the vascular wall. The pattern of shear stress is also well known to be a critical factor in the development of atherosclerosis with laminar shear stress being atheroprotective and disturbed shear stress being pro-atherogenic. While we have a detailed understanding of the various intermediate cell signaling pathways, the receptors that first translate the mechanical stimulus into chemical mediators are not completely understood. Mechanosensitive ion channels are critical to the response to shear and regulate shear-induced cell signaling thereby controlling the production of vasoactive mediators. These channels are among the earliest activated signaling components to shear and have been linked to shear-induced vasodilation through promoting nitric oxide production (e.g., inwardly rectifying K+ [Kir] and transient receptor potential [TRP] channels) and endothelium hyperpolarizing factor (e.g., Kir and calcium-activated K+ [KCa] channels) and shear-induced vasoconstriction through an undetermined mechanism that involves piezo channels. Understanding the biophysical mechanism by which these channels are activated by shear forces (i.e., directly or through a primary mechano-receptor) could provide potential new targets to resolve the pathophysiology associated with endothelial dysfunction and atherogenesis. It is still a major challenge to record flow-induced activation of ion channels in real time using electrophysiology. The standard methods to expose cells to well-defined shear stress, such as the cone and plate rheometer and closed parallel plate flow chamber do not allow real time study of ion channel activation. The goal of this protocol is to describe a modified parallel plate flow chamber that allows real time electrophysiological recording of mechanosensitive ion channels under well-defined shear stress.

The goal of this protocol is to describe a modified parallel plate flow chamber for use in investigating real time activation of mechanosensitive ion channels by shear stress.

Grabaciones electrofisiológicas de corrientes de iones de una sola célula bajo estrés de cizallamiento bien definido

Fluid shear stress is well known to play a major role in endothelial function. In most vascular beds, elevated shear stress from acute increases in blood ...

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

Visualización cinética de interacciones bacterianas entre especies de una sola célula

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...