This work was supported by the Max Planck Society.

1. Preparation of Electro-competent V. parahaemolyticus Cells and Electroporation of Plasmid DNA into Swimmer Cells
NOTE: The following section of the protocol allows for the preparation of electro-competent cells and the subsequent electroporation of plasmid DNA into swimmer cells. This step is important if fluorescent proteins are being ectopically expressed from a plasmid.
<ol>
	<li>Streak out V. parahaemolyticus cells from a -80 &#176;C glycerol stock onto a fresh LB agar plate containing 100 &#181;g/mL ampicillin and incubate overnight at 37 &#176;C.</li>
	<li>The following day, inoculate 200 mL of LB medium with a single colony of V. parahaemolyticus and incubate it at 37 &#176;C under shaking conditions until an OD600 of 1.0 is reached. It usually takes 5-6 h of incubation for the cell culture to reach the desired optical density.</li>
	<li>Immediately transfer the cells onto ice and perform all further steps on ice and in 4 &#176;C pre-cooled centrifuges.</li>
	<li>Harvest the cells at 4 &#176;C for 10 min at 2,000 x g.</li>
	<li>Discard the supernatant by decantation and re-suspend the cell pellet in 25 mL of ice-cold 273 mM sucrose solution (pH 7.4, buffered with KOH).</li>
	<li>Harvest the cells again at 4 &#176;C for 10 min at 2000 x g. Repeat two additional times.</li>
	<li>Re-suspend the washed cell pellet in 400 &#181;L of ice-cold 273 mM sucrose solution. Subsequently, add glycerol to a final concentration of 15% vol/vol. The cells are now ready for electroporation, but can also be frozen in aliquots of 70 &#181;L at -80 &#176;C for later use.</li>
	<li>For the electroporation, mix a 70 &#181;L aliquot of electro-competent cells with 100 - 1,000 ng of plasmid DNA and transfer the mixture into an ice-cold electroporation cuvette (0.2 cm electrode gap). Perform electroporation with the following settings: 25 &#181;F, 2400 V and 200 &#937;.</li>
	<li>Suspend the cells in the electroporation cuvette in 600 &#181;L of LB broth, transfer to a 2 mL tube and incubate while shaking at 37 &#176;C for 3 h.</li>
	<li>Spin down the cells at 2,000 x g for 5 min and re-suspend the pellet in 100 &#181;L of LB broth. Spread the cell-suspension onto selective LB agar plates containing the necessary antibiotics and incubate at 37 &#176;C overnight.</li>
	<li>The following day, check the plates for colony formation. Colonies that grow carry the correct antibiotic resistance gene encoded on the plasmid and can now be used for further experiments.</li>
</ol>
2. Induction of Swarmer Cell Differentiation
NOTE: The following steps describe how to induce differentiation of V. parahaemolyticus into its swarmer cell life-cycle and how to stimulate proliferation on solid surfaces.
<ol>
	<li>Suspend 4 g of Heart Infusion (HI) Agar in 100 mL ddH2O in order to prepare the swarming plates.</li>
	<li>Carefully boil the HI agar solution in the microwave and shake the bottle from time-to-time until the powder is dissolved. Autoclave at 121 &#176;C for 20 min.</li>
	<li>After autoclaving, cool down the HI agar to 60-65 &#176;C. Add 2,2&#180;-Bipyridyl to a final concentration of 50 &#181;M (stock solution of 50 mM in 100% ethanol). Add CaCl2 to a final concentration of 4 mM (stock solution of 4 M in ddH2O). If necessary, add antibiotics into the solution to maintain plasmids. 
	NOTE: If planning to use an inducible plasmid, add inducing agent to its final concentration. 
	CAUTION: 2,2&#180;-Bipyridyl: Acute toxicity (oral, dermal, inhalation). Use gloves and safety goggles when handling this chemical.</li>
	<li>Pour 30 - 35 mL of the final HI agar solution into a round 150 mm Petri dish and let the agar solidify.</li>
	<li>Right before spotting the cell culture, dry the agar plate at 37 &#176;C for at least 10 min (or until any liquid residues have disappeared, but no longer) up-right with an open lid. 
	NOTE: Drying times highly depend on the incubator used and heavily dried plates will not permit swarming of V. parahaemolyticus. (This step is very important!) Plates must be used fresh and should not be older than 2 - 3 h. Old plates will be too dry to stimulate swarming.</li>
	<li>Inoculate a small amount of cells from the edge of a single colony into 5 mL of LB broth. Incubate the culture shaking at 37 &#176;C until OD600 = 0.8 is reached, it usually takes around 2-3 hours. After that, the cells are ready to be spotted onto HI swarming agar plates.</li>
	<li>Spot 1 &#181;L of cell culture at the center of a HI agar swarming plate (Figure 1A).</li>
	<li>Let the spot dry (Figure 1B).</li>
	<li>Seal the plate with clear plastic tape and make sure that it is firmly attached, otherwise it might detach during overnight incubation (Figure 1C). 
	NOTE: It is very important that the seal is perfect. Avoid the formation of air-bubbles and folds to the tape when it is applied.</li>
	<li>Incubate the plate at 24 &#176;C overnight. This will result in the formation of a V. parahaemolyticus swarm-colony with swarm-flares extending from the periphery of the swarm-colony (Figure 2).</li>
</ol>
3. Preparation of V. parahaemolyticus Swarmer Cells for Fluorescence Microscopy
NOTE: The following protocol describes how to prepare microscopy agarose slides and the subsequent imprinting of swarming flares onto an agarose pad to obtain single swarmer cells for microscopy.
<ol>
	<li>To prepare agarose slides for microscopy, add 1 g of agarose to 100 mL of a 20% vol/vol PBS and 10% vol/vol LB broth solution. Carefully boil the solution in the microwave to dissolve the agarose and let it cool down to 65-70 &#176;C while mixing with a magnetic stirrer.</li>
	<li>Place a clean microscope slide onto a perfectly level section of a working bench. Cover both ends of the slide with two layers of general-purpose laboratory labeling tape, fixing the slide to the working bench. The distance between the two pieces of tape should be around 3 cm. 
	NOTE: The two layers of tape create a very small elevation, which will determine the height of the agarose pad. It is important that the working space is level to make sure cells will be in the same plane for microscopy analysis.</li>
	<li>Pipet ~ 250 &#181;L of the previously prepared agarose solution onto the non-covered glass surface.</li>
	<li>Now place a second microscope slide in the same orientation as the bottom one on-top and slightly press it down, so that residual agarose can flow out to the sides.</li>
	<li>Let the agarose solidify 1-2 min and then slowly pull the top glass slide off the bottom one in a horizontal movement.</li>
	<li>Using a scalpel, cut off any residual agarose that is attached to the sides of the microscope slide and slowly remove the tape from the ends of the microscope slide. 
	NOTE: The agarose slide is now ready and should be used as soon as possible as it will slowly begin to dry out.</li>
	<li>To perform microscopy on swarmer cells, cut out a ~3 mm x 10 mm piece of swarming agar from the most outer edge of a swarming colony (Figure 1D). It is very important not to break the tape seal of the swarm-plate until immediately before transfer of swarmer cells to the microscope slide. Once the tape is removed, swarming V. parahaemolyticus cells initiate differentiation into swimmer cells. 
	NOTE: Make sure to direct the cuts from the outside of the swarming colony edge to the inside of the colony (Figure 1D). This way you can prevent &#34;swimmer contamination&#34; into the edge of the swarming colony.</li>
	<li>Transfer the piece of swarming agar onto a microscope agarose slide with the cells facing the agarose pad (Figure 1E). This imprints the swarming cells onto the agarose pad.</li>
	<li>After waiting ~30 s, remove the agar piece carefully from the agarose pad (Figure 1F).</li>
	<li>Place a glass coverslip onto the agarose pad where the cells were imprinted. The cells are now ready for microscopy. 
	NOTE: It is important to image swarmer cells immediately, since swarmer cells slowly initiate differentiation to the swimmer state.</li>
</ol>
4. Preparation of V. parahaemolyticus Swimmer Cell-cultures For Fluorescence Microscopy
NOTE: This section of the protocol describes how to prepare a culture of V. parahaemolyticus to perform fluorescence microscopy on the swimmer cell-type.
<ol>
	<li>Inoculate a small amount of cells from the edge of a single colony into 5 mL of LB broth. Incubate the culture shaking at 37 &#176;C for 1 h or until slight cell-growth becomes visible.</li>
	<li>At this stage, if needed, add inducing agent for expression of proteins of interest. In this example, L-arabinose was added to a final concentration of 0.2% (w/vol) in order to induce expression of YFP-CheW9.</li>
	<li>Incubate the culture shaking for an additional 2 h at 37 &#176;C.</li>
	<li>Follow steps 3.1-3.7 to prepare microscopy agarose slides.</li>
	<li>Spot 1 &#181;L of cell culture at the center of the agarose pad and let the spot incubate until it is dry.</li>
	<li>Place a glass coverslip onto the agarose pad where the cells were spotted. The cells are now ready to be imaged under the fluorescence microscope.</li>
</ol>
5. Image Analysis
This part of the protocol describes a workflow for image analysis, particularly how to extract fluorescence data of single cells for demographic analysis.
<ol>
	<li>Before beginning the image analysis make sure that all microscopy images are saved in 16bit TIF format.</li>
	<li>Load the DIC (or phase contrast) and the corresponding fluorescent channel images into the software.</li>
	<li>Generate an overlay image of both channels by selecting &#34;Display/Overlay images&#34;. Set the number of pictures (&#34;# Images:&#34;) to two, and select the DIC image in the first channel and the fluorescent image in the second channel.</li>
	<li>Select the &#34;Multi-Line&#34; tool from the toolbar and mark cells from one pole to the other through the middle of the cell. In order to get a list of all generated lines (line objects) open &#34;Measure/Region Measurements&#34;. Configure the region measurements to only display the region label, distance, and average intensity.</li>
	<li>Calibrate the pixel size under &#34;Measure/Calibrate Distances&#34; to the pixel size that is specific for your microscope setup and click &#34;Apply To All Open Images&#34;.</li>
	<li>Transfer all regions (line objects) to the fluorescent channel image. Under &#34;Regions/Transfer Regions&#34; select the source image (the overlay image) and the destination image (the fluorescent channel image). Select &#34;All Regions&#34; and press &#34;OK&#34;. 
	NOTE: To be able to later re-produce the exact lines (line objects, regions, ROI), open &#34;Regions/Save Regions&#34; and save the regions.</li>
	<li>Export all region measurements into a spreadsheet by opening &#34;Measure/Region Measurements&#34; and pressing &#34;Open Log&#34;. Confirm to open and export the data. Once the spreadsheet is opened in the background, finally export the data by pressing &#34;F9: Log Data&#34;.</li>
	<li>To determine the position of fluorescent foci along the cell, open &#34;Measure/Linescan&#34;. Set &#34;Linescan Width&#34; to a value so that the whole width of a cell is covered by the region that you previously created. Right-click into the line-scan graph and select &#34;Show Graph Data&#34;. By left-clicking and dragging the mouse-courser along the line-scan to the point of interest, the &#34;Graph Data&#34; window will display the corresponding pixel/distance between the two points. Select the blue highlighted row and then copy the content. 
	NOTE: The entire line-scan intensity profile can be logged to a spreadsheet by pressing &#34;Log Data&#34; in the &#34;Linescan&#34; window. The extracted data can now be used for visualization in graphs or further statistical analysis. Localization patterns over the cell cycle can be easily visualized in a demographic representation of the cell length and the fluorescent intensities along the cell body. These representations can be obtained using the same ROI&#39;s previously selected. The following steps will give an example of how to generate demographics similar to the ones shown in Figures 3C and 4C.
	<ol>
		<li>In Fiji/ImageJ load region markings (.rgn) previously created in the software (step 5.6) using the &#34;Metamorph nd &#38; ROI files importer&#34;. Alternatively, regions of interest (ROIs) of the cells to be analyzed can be created directly in Fiji/ImageJ using the build-in &#34;Segmented Line&#34; tool.</li>
		<li>Open &#34;Analyze/Tools/ROI manager&#34; and mark all active list entries&#34;.</li>
		<li>Afterward, click &#34;More/Multi Plot&#34; and then select &#34;List&#34; in the new &#34;Multi Plot&#34; window. Click on a table entry, then select and &#34;copy all&#34;. Subsequently, paste into a new spreadsheet. 
		NOTE: Make sure that the data of the longest cell (two corresponding X and Y columns with the most number of rows) is the last one to the right in the spreadsheet.</li>
		<li>Save the spreadsheet as a &#34;CSV (Comma delimited) (*.csv)&#34;.</li>
		<li>Download the R scripts from here &#34;https://github.com/ta-cameron/Cell-Profiles&#8221;10. The scripts sort cells by length and normalize the fluorescent intensity profiles of all cells as an average of each cell&#8217;s fluorescence.</li>
		<li>Run the entire script &#34;cell profiles function.R&#34; in R [version 3.0.1;11].</li>
		<li>Edit the &#34;example plots.R&#34; script in line 30 such that the file path refers to the folder where the previously generated .csv file is saved. Additionally, change the file name &#34;profile.csv&#34; in line 31 to the file name chosen for .csv file generated in step 5.9.4. Afterwards, run &#34;example plots.R&#34; and follow the documentation from &#34;https://github.com/ta-cameron/Cell-Profiles&#8221; as well as the comments from the script file to generate demographic analysis.</li>
	</ol>
	</li>
</ol>

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No conflicts of interest declared.

This paper reports a method for microscopy imaging of V. parahaemolyticus swarmer cells, followed by downstream analyses intended to resolve and quantify the subcellular localization and other features of the studied fluorescently labeled proteins. Although several tools exist for microscopy image processing and analysis14,15,16,17,18, the analysis of...

Bacteria constantly experience changes to their external environment and have developed several techniques to change and adapt their behavior accordingly. One such mechanism involves differentiation into distinct cell types that better complement the altered environment. Differentiation often involves major changes in the regulation of the cell cycle, cell morphology, and the spatiotemporal organization of the cells. One organism that can undergo differentiation is Vibrio parahaemolyticus. V. parahaemolyticus belongs to the Vibrionaceae, whichis a family of proteobacteria that usually inhabit fresh or salt water. Vibrionaceae are widely distributed in the environment and include several species that cause intestinal tract infections in humans, also including Vibrio cholerae.V. parahaemolyticus is a dimorphic organism and is able to differentiate into two distinct cell types as a response to accommodate changes to its external milieu. In aqueous environments, it exists as a short rod-shaped swimmer cell with a single polar flagellum positioned at the old cell pole. Upon surface contact, differentiation into a swarmer cell is triggered. Swarmer cell differentiation involves two major changes: swarmer cell morphogenesis through inhibition of cell division, and the induction of a second flagella system. This results in the formation of a peritrichous and highly elongated rod-shaped swarmer cell, which can either continue the swarmer life-style, where division events results in progeny swarmer cells, or alternatively differentiate back into swimmer cells.
Several factors have been reported to induce or influence swarmer differentiation. The primary stimulus appears to be driven by mechanosensing where V. parahaemolyticus uses the polar flagellum as a tactile sensor that detects inhibition of the rotation upon surface contact, but several other factors are involved as well1. In the lab, rotation of the polar flagellum can be artificially inhibited by addition of phenamil, which blocks the sodium-channel driven flagellar rotation, thereby inducing swarmer differentiation2. Furthermore, in an earlier study, Vibrio alginolyticus was induced to swarm on solid media when cells were propagated on growth medium in a Petri dish sealed with clear plastic tape. Alkali-saturated filter paper prevented swarming under these conditions, hence suggesting that one or more volatile acids might be involved in induction of swarming. Thus, sealing the Petri dish with plastic tape likely allowed for the accumulation of volatile acids, formed as by-products of cellular metabolism, within the head space of the plate. The same effect was achieved when H2O2 was added to the growth medium in un-sealed Petri dishes in order to artificially produce volatile acids by hydrolysing media components3,4,5. Nevertheless, the identity of such volatile acids remains unknown. Moreover, it has been shown that excess availability of calcium6 and iron-limitation7 both enhance swarmer differentiation and proliferation over solid surfaces. Cells can be starved for iron by adding the compound 2,2&#180;-Bipyridyl to the growth medium, which has been shown to influence swarmer differentiation8. The factors known to regulate differentiation are now implemented in the design of a protocol that reproducibly induces V. parahaemolyticus differentiation into swarmer cells and their proliferation on solid agar surfaces.
V. parahaemolyticus differentiation involves major changes in the regulation of cell division, cellular morphology, and the positioning of macromolecular machines such as flagella and chemotaxis apparatuses &#8211; processes that all require the specific localization of proteins in accordance with the cell cycle. Thus, the ability to study the intracellular localization of such proteins is essential to the understanding of the aforementioned cellular processes. In order to perform such studies fluorescence microscopy on single cells is required. This can be particularly challenging when imaging cells that exist within dense bacterial populations, as is the case for swarmer cells. There have been attempts to induce swarmer differentiation in liquid media, which could potentially generate single cells for microscopy studies. And although on a transcriptional level these cells have partially induced the swarmer-differentiation program, they do not undergo the same distinct morphological changes as fully differentiated swarmer cells grown on solid medium8. This paper offers a robust and reproducible protocol of a method to induce swarmer cell differentiation on an agar surface. The protocol produces a population of easily accessible swarmer cells readily available for single cell microscopy and subsequent analysis. Furthermore, the protocol enables localization studies of fluorescently labeled proteins in both the swimmer and swarmer cell-types (sections 1, 2, 3, 4). Additionally, the protocol describes the subsequent workflow on how to process and analyze the generated data from fluorescence microscopy experiments, which allows for demographic analysis of bacterial societies (section 5).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Media components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LB-medium</td>
			<td>Roth</td>
			<td>X968.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Agar, granulated</td>
			<td>BD</td>
			<td>214510</td>
			<td>For preparation of LB agar with LB-medium</td>
		</tr>
		<tr>
			<td>Difco Heart Infusion Agar</td>
			<td>BD</td>
			<td>244400</td>
			<td>For preparation of HI agar swarming plates</td>
		</tr>
		<tr>
			<td>Agarose NEEO, ultra quality</td>
			<td>Roth</td>
			<td>2267.3</td>
			<td>For preparation of microscopy agarose pads</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Additives</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-arabinose</td>
			<td>Roth</td>
			<td>5118.3</td>
			<td>Used to induce pBAD derivatives</td>
		</tr>
		<tr>
			<td>2,2`-Bipyridyl</td>
			<td>Sigma Aldrich</td>
			<td>D216305-25G</td>
			<td>For preparation of HI agar swarming plates</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Roth</td>
			<td>5239.1</td>
			<td>For preparation of HI agar swarming plates</td>
		</tr>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>Roth</td>
			<td>K029.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Roth</td>
			<td>3886.3</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS buffer</td>
			<td>-</td>
			<td>-</td>
			<td>Standard recipe for Phosphate-buffered saline</td>
		</tr>
		<tr>
			<td>D(+) Sucrose</td>
			<td>Roth</td>
			<td>4621.1</td>
			<td>For preparation of electrocompetent cells</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Roth</td>
			<td>3783.5</td>
			<td>For preparation of electrocompetent cells</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Hardware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 150x20mm with cams</td>
			<td>Sarstedt</td>
			<td>82.1184.500</td>
			<td>For preparation of HI agar swarming plates</td>
		</tr>
		<tr>
			<td>kelvitron t (B 6420)</td>
			<td>Heraeus</td>
			<td>-</td>
			<td>Used for drying HI agar swarming plates</td>
		</tr>
		<tr>
			<td>Gene Pulser Xcell Microbial System</td>
			<td>BioRad</td>
			<td>1652662</td>
			<td>Used for elctroporation of plasmid DNA</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MetaMorph Offline (version 7.7.5.0)</td>
			<td>Molecular Devices</td>
			<td>-</td>
			<td>Used for microscopy image analysis</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td>-</td>
			<td>Used for microscopy data analsis</td>
		</tr>
	</tbody>
</table>

induction of cellular differentiation and single cell imaging of vibrio parahaemolyticus swimmer and swarmer cells

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ability to obtain single cells for fluorescence microscopy, which can be particularly challenging when imaging cells that exist within bacterial communities. For example, the human pathogen Vibrio parahaemolyticus exists as short rod-shaped swimmer cells in liquid conditions that upon surface contact differentiate into a subpopulation of highly elongated swarmer cells specialized for growth on solid surfaces. This paper presents a method to perform single cell fluorescence microscopy analysis of V. parahaemolyticus in its two differential states. This protocol very reproducibly induces differentiation of V. parahaemolyticus into a swarmer cell life-cycle and facilitates their proliferation over solid surfaces. The method produces flares of differentiated swarmer cells extending from the edge of the swarm-colony. Notably, at the very tip of the swarm-flares, swarmer cells exist in a single layer of cells, which allows for their easy transfer to a microscope slide and subsequent fluorescence microscopy imaging of single cells. Additionally, the workflow of image analysis for demographic representation of bacterial societies is presented. As a proof of principle, the analysis of the intracellular localization of chemotaxis signaling arrays in swimmer and swarmer cells of V. parahaemolyticus is described.

Induction of differentiation and generation of swarming colonies
Figure 1 provides a schema of the important steps involved in producing swarming colonies of V. parahaemolyticus (Figure 1A-C). A swimmer culture was spotted on swarm-agar and incubated at 24 oC, inducing swarmer differentiation and proliferation over the solid agar surface. A representative stereo-microscopy image...

Watch this Scientific Journal Video about Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells at JoVE.com

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

This protocol enables single cell microscopy of the differentially distinct Vibrio parahaemolyticus swimmer and swarmer cells. The method produces a population of swarmer cells easily available for single cell analysis and covers preparation of cell cultures, induction of swarmer differentiation, sample preparation, and image analysis.

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ...

induction-cellular-differentiation-single-cell-imaging-vibrio

Research

JoVE Journal

Immunology and Infection

9.3K Views.  Max Planck Institute for Terrestrial Microbiology. This protocol enables single cell microscopy of the differentially distinct Vibrio parahaemolyticus swimmer and swarmer cells. The method produces a population of swarmer cells easily available for single cell analysis and covers preparation of cell cultures, induction of swarmer differentiation, sample preparation, and image analysis.

Video: Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &alpha;-actinin by Confocal Imaging

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an effective invasin for human epithelial and endothelial cells. We have identified endothelial surface-located integrins as major receptors for Opc, a process which requires Opc to first bind to integrin ligands such as vitronectin and via these to the cell-expressed receptors1. This process leads to bacterial invasion of endothelial cells2. More recently, we observed an interaction of Opc with a 100kDa protein found in whole cell lysates of human cells3. We initially observed this interaction when host cell proteins separated by electrophoresis and blotted on to nitrocellulose were overlaid with Opc-expressing Nm. The interaction was direct and did not involve intermediate molecules. By mass spectrometry, we established the identity of the protein as &alpha;-actinin. As no surface expressed &alpha;-actinin was found on any of the eight cell lines examined, and as Opc interactions with endothelial cells in the presence of serum lead to bacterial entry into the target cells, we examined the possibility of the two proteins interacting intracellularly. For this, cultured human brain microvascular endothelial cells (HBMECs) were infected with Opc-expressing Nm for extended periods and the locations of internalised bacteria and &alpha;-actinin were examined by confocal microscopy. We observed time-dependent increase in colocalisation of Nm with the cytoskeletal protein, which was considerable after an eight hour period of bacterial internalisation. In addition, the use of quantitative imaging software enabled us to obtain a relative measure of the colocalisation of Nm with &alpha;-actinin and other cytoskeletal proteins. Here we present a protocol for visualisation and quantification of the colocalisation of the bacterium with intracellular proteins after bacterial entry into human endothelial cells, although the procedure is also applicable to human epithelial cells.

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &#945;-actinin by Confocal Imaging

Neisseria meningitidis (Nm), a gram negative human-specific respiratory pathogen, can bind to human &alpha;-actinin. Here we present a protocol for visualisation of colocalisation of the bacterium with intracellular &alpha;-actinin after bacterial entry into human brain microvascular endothelial cells (HBMECs).

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an ...

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression of proteins known to respond to metabolic stress was measured in brain-nuclei isolates using Western blotting. This was done to assess whether metabolic stress-induced nutrient insufficiency in the body triggered neuronal stress. We have previously demonstrated that nutrient insufficiency in neonates elicits metabolic stress in the gut. Furthermore, colostrum oxytocin modulates cellular stress response, inflammation, and autophagy markers in newborn rat gut villi prior to and after first feed. Signaling protein markers associated with the endoplasmic reticulum stress [ER chaperone binding immunoglobulin protein (BiP), eukaryotic translation initiation factor 2A (eIF2a), and eIF2a kinase protein kinase R (p-PKR)], as well as two inflammation-signaling proteins [nuclear factor-&#954;B (NF-kB) and inhibitor &#954;B (IkB)], were measured in newborn brain nuclei [nucleus of the solitary tract (NTS), paraventricular nucleus (PVN), supra-optic nucleus (SON), cortex (CX), striatum nuclei (STR), and medial preoptic nucleus (MPO)] before the first feed (unprimed by colostrum) and after the start of nursing (primed by colostrum). Expression of BiP/GRP78 and p-eIF2a were upregulated in unprimed and downregulated in primed NTS tissue. NF-kB was retained (high) in the CX, STR, and MPO cytoplasm, whereas NF-kB was lower and unchanged in NTS, PVN, and SON in both conditions. The collective BiP and p-eIF2 findings are consistent with a stress response. eIf2a was phosphorylated by dsRNA dependent kinase (p-PKR) in the SON, CX, STR, and MPO. However, in the NTS (and to a lesser extent in PVN), eIf2a was phosphorylated by another kinase, general control nonderepressible-2 kinase (GCN2). The stress-modulating mechanisms previously observed in newborn gut enterocytes appear to be mirrored in some OTR-rich brain regions. The NTS and PVN may utilize a different phosphorylation mechanism (under nutrient deficiency) from other regions and be refractory to the impact of nutrient insufficiency. Collectively, this data suggests that brain responses to nutrient insufficiency stress are offset by signaling from colostrum-primed enterocytes.

Here, we present a protocol to isolate brain nuclei in the neonatal rat brain in conjunction with first colostrum feeding. This technique allows the study of nutrient insufficiency stress in the brain as modulated by enterocyte signaling.

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression ...

Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This technique allows for probing interaction strengths and dynamics on a live cell membrane, including those between cells, receptor and ligands, and alongside many other variations. It also works as a mechanism to deliver a physical or biochemical stimulus on single cells in a spatiotemporally controlled manner, thus allowing specific cell activation and subsequent cellular events to be monitored in real-time when combined with live-cell fluorescence imaging. The key step in those AFM-SCFS measurements is AFM-cantilever functionalization, or in other words, attaching a subject of interest to the cantilever. Here, we present methods to modify AFM cantilevers with a single T cell and a single polystyrene bead respectively for immunological studies. The former involves a biocompatible glue that couples single T cells to the tip of a flat cantilever in a solution, while the latter relies on an epoxy glue for single bead adhesion in the air environment. Two immunological applications associated with each cantilever modification are provided as well. The methods described here can be easily adapted to different cell types and solid particles.

We present a protocol to functionalize atomic force microscope (AFM) cantilevers with a single T cell and bead particle for immunological studies. Procedures to probe single-pair T cell-dendritic cell binding by AFM and to monitor the real-time cellular response of macrophages to a single solid particle by AFM with fluorescence imaging are shown.

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This ...