quantitative time-lapse microscopy for assessing extracellular matrix stiffness-dependent bacterial dissemination

Quantitative Time-Lapse Microscopy for Assessing Extracellular Matrix Stiffness-Dependent Bacterial Dissemination

After preparing an overnight culture of L. monocytogenes according to the text protocol, transfer 1 milliliter of the culture into a microcentrifuge tube, and spin it down at 2,000 times g and room temperature for 4 minutes. After using tissue culture-grade PBS to wash the pellet twice, use 1 milliliter of PBS to resuspend the pellet.
Prepare the infection mix by combining 10 or 50 microliters of the bacterial suspension with 1 milliliter of MCDB-131 full medium for a multiplicity of infection, or MOI, of approximately 50 bacteria per host cell or 10 bacteria per host cell. Remove the medium from the wells of the 24-well plates, taking care not to disrupt the hydrogels or the cells.
Use 1 milliliter of MCDB-131 full medium to wash the cells once, then add 1 milliliter of the bacteria to each well. Place the lid on the plates, and wrap them with polyethylene food wrap to avoid leakage. Centrifuge the plates at 2,000 x g for 10 minutes to synchronize the invasion. Then, incubate the cultures at 37 degrees Celsius for 30 minutes. With MCDB-131 full medium, wash the samples 4 times and return them to the tissue culture incubator. After an additional 30 minutes, replace the medium with MCDB-131 full medium supplemented with 20 micrograms per milliliter of gentamicin.
After seeding HMEC-1 cells on PA hydrogels and treating with gentamicin according to the text protocol, incubate the plate for five hours to allow the ActA promoter to turn on and drive the expression of the mTagRFP open reading frame.
Four hours post-infection, mix 1 microliter of 1mg/ml Hoechst dye with 1 milliliter of L15 full medium and add it to each well to stain the nuclei. After incubating the cells for 10 minutes, replace the medium with 1 milliliter of L15 full medium supplemented with 20 micrograms per milliliter of gentamicin.
Image multiple positions every 5 minutes using an autofocus feature to monitor how LM bacteria spread through HMEC-1 monolayers seeded on varying stiffness hydrogels.

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<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide pellets</td>
			<td>Fisher</td>
			<td>S318-500</td>
			<td></td>
		</tr>
		<tr>
			<td>(3-Aminopropyl)triethoxysilane</td>
			<td>Sigma</td>
			<td>A3648</td>
			<td></td>
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		<tr>
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		<tr>
			<td>40% Acrylamide</td>
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		</tr>
		<tr>
			<td>Bis-acrylamide solution (2%w/v)</td>
			<td>Fisher Scientific</td>
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		<tr>
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			<td>Fisher</td>
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			<td>TEMED</td>
			<td>Sigma</td>
			<td>T9281-25ML</td>
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			<td>c1111-100mg</td>
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			<td>Collagen, Type I Solution from rat</td>
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			<td>C3867-1VL</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>J.T. Baker</td>
			<td>9224-01</td>
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		</tr>
		<tr>
			<td>HEPES, Free acid</td>
			<td>J.T. Baker</td>
			<td>4018-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 medium, no phenol red</td>
			<td>Thermofischer</td>
			<td>21083027</td>
			<td></td>
		</tr>
		<tr>
			<td>MCDB 131 Medium, no glutamine</td>
			<td>Life technologies</td>
			<td>10372019</td>
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			<td>Foundation Fetal Bovine Serum, Lot: A37C48A</td>
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			<td>L-Glutamine 200mM</td>
			<td>Fisher</td>
			<td>SH3003401</td>
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		<tr>
			<td>DPBS 1X</td>
			<td>Fisher</td>
			<td>SH30028FS</td>
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		</tr>
		<tr>
			<td>Gentamicin sulfate</td>
			<td>MP biomedicals</td>
			<td>194530</td>
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			<td>Chloramphenicol</td>
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			<td>211059</td>
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			<td>Hoechst 33342, trihydrochloride, trihydrate - 10 mg/ul solution in water</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td></td>
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			<td>0.25% trypsin-EDTA , phenol red</td>
			<td>Thermofischer</td>
			<td>25200056</td>
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			<td>Streptomycin sulfate</td>
			<td>Fisher Scientific</td>
			<td>3810-74-0</td>
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			<td>Disposable lab equipment</td>
			<td></td>
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			<td>12 mm circular glass coverslips</td>
			<td>Fisherbrand</td>
			<td>12-545-81</td>
			<td>No. 1.5 Coverslip | 10 mm Glass Diameter | Uncoated</td>
		</tr>
		<tr>
			<td>Glass bottom 24 well plates</td>
			<td>Mattek</td>
			<td>P24G-1.5-13-F</td>
			<td></td>
		</tr>
		<tr>
			<td>T-25 flasks</td>
			<td>Falcon</td>
			<td>353118</td>
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			<td>50 ml conical tubes</td>
			<td>Falcon</td>
			<td>352070</td>
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			<td>15 ml conicals tubes</td>
			<td>Falcon</td>
			<td>352196</td>
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			<td>Disposable Serological Pipettes (1 ml, 2 ml, 5 ml, 10 ml, 25 ml)</td>
			<td>Falcon</td>
			<td>357551</td>
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		</tr>
		<tr>
			<td>Pasteur Glass Pipettes</td>
			<td>VWR</td>
			<td>14672-380</td>
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		</tr>
		<tr>
			<td>Pipette Tips (1-200 &#956;l, 101-1000 &#956;l)</td>
			<td>Denville</td>
			<td>P1122, P1126</td>
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		</tr>
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			<td>Powder Free Examination Gloves</td>
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			<td>XC-310</td>
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		<tr>
			<td>Cuvettes bacteria</td>
			<td>Sarstedt</td>
			<td>67.746</td>
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			<td>Razors</td>
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			<td>0.2um sterilizng bottles</td>
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			<td>wooden sticks</td>
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			<td>42181501</td>
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		<tr>
			<td>Saran wrap</td>
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			<td>sc-3687</td>
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		</tr>
		<tr>
			<td>Plates bacteria</td>
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			<td>351029</td>
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			<td>Hemacytometer</td>
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			<td>Z359629</td>
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			<td>Bacteria incubator</td>
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			<td>IGS180</td>
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		<tr>
			<td>Tissue culture Incubator</td>
			<td>NuAire</td>
			<td>NU-8700</td>
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		</tr>
		<tr>
			<td>Vacuum chamber/degasser</td>
			<td>Belart</td>
			<td>42025</td>
			<td>37 &#176;C and 5% CO2</td>
		</tr>
		<tr>
			<td>Inverted Nikon Diaphot 200 epifluorescence microscope</td>
			<td>Nikon</td>
			<td>NIKON-DIAPHOT-200</td>
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		</tr>
		<tr>
			<td>Cage Incubator</td>
			<td>Haison</td>
			<td>Custom</td>
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		</tr>
		<tr>
			<td>Pipette Aid</td>
			<td>Drummond</td>
			<td>4-000-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettors (10 &#956;l, 200 &#956;l, 1000ul)</td>
			<td>Gilson</td>
			<td>F144802, F123601, F123602</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Mettler Toledo</td>
			<td>30019028</td>
			<td></td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>FST</td>
			<td>11000-12</td>
			<td></td>
		</tr>
		<tr>
			<td>1 L flask</td>
			<td>Fisherbrand</td>
			<td>FB5011000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave machine</td>
			<td>Amsco</td>
			<td>3021</td>
			<td></td>
		</tr>
		<tr>
			<td>Stir magnet plate</td>
			<td>Bellco</td>
			<td>7760-06000</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnet stirring bars</td>
			<td>Bellco</td>
			<td>1975-00100</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Beckman</td>
			<td>DU 640</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Software (&#956;Manager)</td>
			<td>Open Imaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>Matlab Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flowjo</td>
			<td>FlowJo, LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Automated image analysis software, CellC</td>
			<td>https://sites.google.com/site/cellcsoftware/</td>
			<td></td>
			<td>The software is freely available. Eexecutable files and MATLAB source codes can be obtained at https://sites.google.com/site/cellcsoftware/</td>
		</tr>
	</tbody>
</table>

Source: Bastounis, E. E. et al., <a href="https://app.jove.com/v/57361">A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells.</a> J. Vis. Exp.&#160;(2018)
This video showcases quantitative time-lapse microscopy for evaluating extracellular matrix stiffness-dependent Listeria monocytogenes dissemination through endothelial cells. A softer hydrogel facilitates faster bacterial dissemination through endothelial cells compared to stiffer counterparts, highlighting the influence of matrix stiffness on bacterial dissemination.

<img alt="Figure 1" src="/files/ftp_upload/22006/22006_Figure1.jpg" /> 
Figure 1: Bacterial infection assay of host cells residing on thin two-layered fluorescent bead-embedded polyacrylamide (PA) hydrogels of varying stiffness. A.&#160;Glass coverslips are chemically modified to enable hydrogel attachment.&#160;B. 3.6 &#181;L of PA mixtures are deposited on the glass bottoms.&#160;C. The mixture is covered with a 12-mm circular glas...

1. Manufacturing Thin Two-layered Polyacrylamide (PA) Hydrogels on Multi-Well Plates

	Dissolve ammonium persulfate (APS) in distilled ultrapure water to ...

Take a multi-well plate with adherent human endothelial cell monolayers on collagen-coated polyacrylamide hydrogels of varying stiffness.
Cells on stiffer hydrogels undergo significant cytoskeletal reorganization, increasing intracellular tension, compared to softer hydrogels.
Wash cells with media and add engineered Listeria monocytogenes &#8212; expressing a fluorescent protein under the actin assembly-inducing protein (ActA) promoter.
Centrifuge to facilitate cell-bacteria contact. Incubate to mediate bacterial endocytosis.
Wash with media. Treat with gentamicin to eliminate non-internalized bacteria. Incubate.
Internalized bacteria release pore-forming toxins, disrupting phagosomal membranes. Cytosolic bacteria upregulate ActA and fluorescent protein expression, labeling intracellular bacteria.
ActA proteins induce actin polymerization, forming an actin comet tail propelling the bacteria forward, causing internalization by neighboring cells, and promoting dissemination.
Post-incubation, use dye-containing media to stain cell nuclei. Replace media with media containing gentamicin.
Perform time-lapse microscopy.
Softer hydrogels promote rapid bacteria intercellular spread compared to stiffer hydrogels with increased intracellular tension, suggesting matrix stiffness-dependent bacterial dissemination.

14 Views. Ilościowa mikroskopia poklatkowa do oceny rozsiewu bakterii zależnego od sztywności macierzy zewnątrzkomórkowej

Video: Ilościowa mikroskopia poklatkowa do oceny rozsiewu bakterii zależnego od sztywności macierzy zewnątrzkomórkowej - Experiment

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 ...

JoVE Journal

Immunology and Infection

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, rhizosphere colonization by plant growth-promoting bacteria (PGPB) can increase the health or the development of host plants relative to uncolonized plants. However, in field settings, bacterial treatments with PGPB often do not provide substantial benefits to crops. One explanation is that this may be due to loss of the PGPB during interactions with endogenous soil microbes over the lifespan of the plant. This possibility has been difficult to confirm, since most studies focus on the initial colonization rather than maintenance of PGPB within rhizosphere communities. It is hypothesized here that the assembly, coexistence, and maintenance of bacterial communities are shaped by deterministic features of the rhizosphere microenvironment, and that these interactions may impact PGPB survival in native settings. To study these behaviors, a hydroponic plant-growth assay is optimized using Arabidopsis thaliana to quantify and visualize the spatial distribution of bacteria during initial colonization of plant roots and after transfer to different growth environments. This system&#8217;s reproducibility and utility are then validated with the well-studied PGPB Pseudomonas simiae. To investigate how the presence of multiple bacterial species may affect colonization and maintenance dynamics on the plant root, a model community from three bacterial strains (an Arthrobacter, Curtobacterium, and Microbacterium species) originally isolated from the A. thaliana rhizosphere is constructed. It is shown that the presence of these diverse bacterial species can be measured using this hydroponic plant-maintanence assay, which provides an alternative to sequencing-based bacterial community studies. Future studies using this system may improve the understanding of bacterial behavior in multispecies plant microbiomes over time and in changing environmental conditions.

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Here we describe a hydroponic plant growth assay to quantify species presence and visualize the spatial distribution of bacteria during initial colonization of plant roots and after their transfer into different growth environments.

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, ...

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC).
To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...

Quantitative Time-Lapse Microscopy for Assessing Extracellular Matrix Stiffness-Dependent Bacterial Dissemination

Transkrypcja

Quantitative Time-Lapse Microscopy for Assessing Extracellular Matrix Stiffness-Dependent Bacterial Dissemination