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1. Internalization of Bacteria into Host Cells (Fluorescent Microscopy)
Note: P. gingivalis is labeled with 2&#39;,7&#39;-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester (BCECF-AM). BCECF-AM is a non-fluorescent membrane-permeable dye; its conversion to fluorescein BCECF via the action of intracellular esterases can indicate cell viability. P. gingivalis is labeled with the BCECF-AM dye and then used to infect eukaryotic cells. Following infection, cells are fixed and labeled with DAPI and TRITC-phalloidin. The DAPI stain used to stain the eukaryotic cell nucleus will also label bacterial cell nucleus, which provides a countermeasure to identify non-viable bacteria that cannot metabolically cleave BCECF-AM. Host cells are highlighted with TRITC-phalloidin, a red actin dye.
<ol>
	<li>Autoclave coverslips. Aseptically add coverslips to 12-well plates before seeding endothelial cells at 5 x 104 cells/well. (Prepared day before experiment)</li>
	<li>Have endothelial cells prepared on 18 mm (#1.5 thickness) circular coverslips in 12-well plates as described above.</li>
	<li>Prepare anaerobic bacteria grown to mid-log phase (OD660 = 0.5-0.7).</li>
	<li>Wash bacteria 2x with anaerobic PBS by centrifuging at 5,000 x g and suspending the pellet in PBS at 5-7 x 108 cells/ml.</li>
	<li>Add 20 &#181;l of 0.2 mM BCECF-AM to 2 ml of bacterial suspension (5-7 x 108 cells/ml) to a final concentration of BCECF-AM of 2 &#181;M.</li>
	<li>Incubate at 37 &#176;C for 30 min in the dark.</li>
	<li>Transfer plates with endothelial cells seeded on 18 mm (#1.5 thickness) circular coverslips from tissue culture incubator into the anaerobic chamber. Wash with PBS and exchange with anaerobic VEGF media. 
	Note: Verify that HUVECs are healthy under a light microscope. HUVECS should be ~80% confluent, morphology should be comparable to the manufacturers.</li>
	<li>Centrifuge labeled bacteria at 5,000 x g for 10 min to remove residual BCECF-AM dye. Suspend in 2 ml anaerobic VEGF media.</li>
	<li>Infect host cells with labeled bacteria at MOI of 100:1 (bacteria: host).</li>
	<li>Incubate in an anaerobic chamber at 37 &#176;C for 30 min.</li>
	<li>After infection wash cells with PBS three times and fix in freshly prepared 4.0% paraformaldehyde for 10 min. 
	Note: After fixing cells, experiments can be conducted outside of the anaerobic chamber.</li>
	<li>Wash coverslips with PBS three times.</li>
	<li>Add 1 ml of 0.2% Triton X-100 for 10 min.</li>
	<li>Wash coverslips with PBS three times.</li>
	<li>Add 50 &#181;l of TRITC phalloidin (50 &#181;g/ml) to coverslips for 45 min.</li>
	<li>Wash coverslips three times, remove from the 12-well plate and place on a slide with a soft-set mounting medium containing DAPI. Seal the sides with nail polish. 
	Note: Slides may be stored for a couple months in the dark. Avoid light exposure to prevent photo-bleaching.</li>
	<li>View slides using a confocal microscope.
	<ol>
		<li>Here, use a 34 channel spectral system (32-channel array detector and two side PMT detectors, plus a transmitted light detector) configured around an AxioObserver (inverted) stand with a motorized XY stage. The system has five lasers: blue diode (405 nm), multi-line Argon (458, 488, 514 nm), green diode (561 nm), red HeNe (633 nm) and a 440 nm pulsed laser. Equip a Fluorescence Lifetime Imaging system with 2 hybrid GaAsP detectors (for FRET-FLIM).</li>
		<li>Detect the fluorescence from DAPI and TRITC in one channel using a dual-band filter with excitation wavelengths of 340-380 nm and 540-560 nm, and an emission filter of 435-485 nm and 570-590 nm respectively. Detect the fluorescence from BCECF-AM using a filter with excitation wavelength of 440-500 nm and an emission filter of 510-590 nm. 
		Note: Controls for BCECF-AM should be done on every bacterial strain being studied to ensure proper labeling of viable bacteria. First validate that nonviable bacteria are DAPI-positive and BCECF-negative. Second, ensure that live bacteria can metabolize BCECF-AM into fluorescein BCECF. Variable concentrations of the bacteria or BCECF-AM dye may need to be tested for optimal labeling.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Vinyl Anaerobic Chamber-Type B</td>
			<td>Coy Laboratory Products</td>
			<td></td>
			<td>Model 2000 incubator</td>
		</tr>
		<tr>
			<td>TSA II Trypticase Soy Agar w/5% Sheep Blood</td>
			<td>BBL</td>
			<td>221261</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Umbilical Vein Endothelial Cells 10-donor Pool</td>
			<td>LifeLine Technology</td>
			<td>FC-0044</td>
			<td></td>
		</tr>
		<tr>
			<td>VascuLife VEGF Medium Complete Kit</td>
			<td>LifeLine Technology</td>
			<td>LL-0003</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypKit</td>
			<td>LifeLine</td>
			<td>LL-0013</td>
			<td></td>
		</tr>
		<tr>
			<td>BCECF-AM</td>
			<td>LifeTechnologies</td>
			<td>B1150</td>
			<td></td>
		</tr>
		<tr>
			<td>TRITC Phalloidin</td>
			<td>Sigma-Aldrich</td>
			<td>P1951</td>
			<td></td>
		</tr>
		<tr>
			<td>18 mm Circular Coverslips</td>
			<td>Electron Microscopy Sciences</td>
			<td>72222-01</td>
			<td></td>
		</tr>
		<tr>
			<td>VectaShield Mounting Medium with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of internalized anaerobic bacteria within host endothelial cells

Source: Wunsch, C. M. et. al., <a href="https://app.jove.com/v/53408">Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells.</a> J. Vis. Exp. (2015)
In this video, we demonstrate a procedure to visualize internalized Porphyromonas gingivalis, an anaerobic pathogenic bacterium, within human endothelial cells using confocal microscopy. The bacteria are stained with a fluorescent dye while the host endothelial cell actin cytoskeleton is labeled with a different fluorescent dye. The internalized Porphyromonas gingivalis inside endothelial cells are visualized using confocal microscopy.

Visualization of Internalized Anaerobic Bacteria within Host Endothelial Cells

1. Internalization of Bacteria into Host Cells (Fluorescent Microscopy)
Note: P. gingivalis is labeled with ...

Begin with a suspension of actively growing Porphyromonas gingivalis, anaerobic pathogenic bacteria.
Add BCECF-AM, a non-fluorescent, cell-permeable dye.
Active cytoplasmic esterases hydrolyze the dye into a fluorescent, cell-impermeable form, staining the bacteria.
Centrifuge. Resuspend the bacteria in anaerobic media.
Add the labeled bacteria to multi-well plate wells containing coverslips with adhered human endothelial cells.
Incubate in an anaerobic chamber.
The bacterial fimbriae bind to the endothelial cell surface receptors, initiating intracellular signaling cascades.
This leads to actin rearrangement and bacterial internalization within phagosomes.
Post-incubation, wash coverslips with buffer to remove non-internalized bacteria.
Fix the cells with paraformaldehyde to preserve the cellular structures. Permeabilize the cells with a non-ionic detergent to access intracellular targets.
Add fluorescently labeled phalloidin, which binds to the actin filaments.
Place the coverslip over a mounting medium containing DAPI, which stains the DNA.
Observe the fluorescent Porphyromonas gingivalis within endothelial cells with fluorescent actin, confirming bacterial internalization.

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Imaging and Visualization Techniques

68 Views. Source: Wunsch, C. M. et. al., Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells. J. Vis. Exp. (2015) In this video, we demonstrate a procedure to visualize internalized Porphyromonas gingivalis, an anaerobic pathogenic bacterium, within human endothelial cells using confocal microscopy. The bacteria are stained with a fluorescent dye while the host endothelial cell actin cytoskeleton is labeled with a different fluorescent dye. The int...

Video: Visualization of Internalized Anaerobic Bacteria within Host Endothelial Cells - Concept

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.

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

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Immunology and Infection

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3).
Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

The mechanism associated with phagocytosis in Leishmania infection remains poorly understood. Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells.

Visualization of Internalized Anaerobic Bacteria within Host Endothelial Cells

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