Name: induction of cellular differentiation and single cell imaging of vibrio parahaemolyticus swimmer and swarmer cells
Uploaded: 2017-05-15T15:00:00
Description: Watch this Scientific Journal Video about Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells at JoVE.com

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 ag...

<ol>
	<li>McCarter, L., Hilmen, M., Silverman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flagellar+dynamometer+controls+swarmer+cell+differentiation+of+V.+parahaemolyticus.">Flagellar dynamometer controls swarmer cell differentiation of V. parahaemolyticus.</a> Cell. 54 (3), 345-351 (1988).</li><li>Kawagishi, I., Imagawa, M., Imae, Y., McCarter, L., Homma, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sodium-driven+polar+flagellar+motor+of+marine+Vibrio+as+the+mechanosensor+that+regulates+lateral+flagellar+expression.">The sodium-driven polar flagellar motor of marine Vibrio as the mechanosensor that regulates lateral flagellar expression.</a> Mol. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R:+A+Language+and+Environment+for+Statistical+Computing.">R: A Language and Environment for Statistical Computing.</a> R Foundation for Statistical Computing. 1, 2673 (2008).</li><li>Ringgaard, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ParP+prevents+dissociation+of+CheA+from+chemotactic+signaling+arrays+and+tethers+them+to+a+polar+anchor.">ParP prevents dissociation of CheA from chemotactic signaling arrays and tethers them to a polar anchor.</a> Proc Natl Acad Sci U S A. 111 (2), E255-E264 (2014).</li><li>Ringgaard, S., Schirner, K., Davis, B. M., Waldor, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+family+of+ParA-like+ATPases+promotes+cell+pole+maturation+by+facilitating+polar+localization+of+chemotaxis+proteins.">A family of ParA-like ATPases promotes cell pole maturation by facilitating polar localization of chemotaxis proteins.</a> Genes Dev. 25, 1544-1555 (2011).</li><li>Vischer, N. O. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+age+dependent+concentration+of+Escherichia+coli+divisome+proteins+analyzed+with+ImageJ+and+ObjectJ.">Cell age dependent concentration of Escherichia coli divisome proteins analyzed with ImageJ and ObjectJ.</a> Front Microbiol. 6 (JUN), (2015).</li><li>Schneider, C. a., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat. Methods. 9 (7), 671-675 (2012).</li><li>Sliusarenko, O., Heinritz, J., Emonet, T., Jacobs-Wagner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput,+subpixel+precision+analysis+of+bacterial+morphogenesis+and+intracellular+spatio-temporal+dynamics.">High-throughput, subpixel precision analysis of bacterial morphogenesis and intracellular spatio-temporal dynamics.</a> Mol. Microbiol. 80 (3), 612-627 (2011).</li><li>Ducret, A., Quardokus, E. M., Brun, Y. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicrobeJ,+a+tool+for+high+throughput+bacterial+cell+detection+and+quantitative+analysis.">MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis.</a> Nat. Microbiol. 1, 16077 (2016).</li><li>Carpenter, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CellProfiler:+image+analysis+software+for+identifying+and+quantifying+cell+phenotypes.">CellProfiler: image analysis software for identifying and quantifying cell phenotypes.</a> Genome Biol. 7 (10), R100 (2006).</li><li>Morales-Soto, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation,+Imaging,+and+Quantification+of+Bacterial+Surface+Motility+Assays.">Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays.</a> J. Vis. Exp. (98), e52338 (2015).</li><li>Paintdakhi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oufti:+An+integrated+software+package+for+high-accuracy,+high-throughput+quantitative+microscopy+analysis.">Oufti: An integrated software package for high-accuracy, high-throughput quantitative microscopy analysis.</a> Mol. Microbiol. 99 (4), 767-777 (2016).</li><li>de Jong, I. G., Beilharz, K., Kuipers, O. P., Veening, J. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Cell+Imaging+of+Bacillus+subtilis+and+Streptococcus+pneumoniae+using+Automated+Time-lapse+Microscopy.">Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy.</a> J. Vis. Exp. (53), e1-e6 (2011).</li></ol>

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 ...

The overall goal of this method is to induce cellular differentiation of the bacterium Vibrio parahaemolyticus and the formation of a population of differentiated swarmer cells easily available for single cell analysis by fluorescence microscopy. This method will help scientists understand how bacteria regulate the intracellular organization during cellular differentiation and morphogenesis. Since imaging cells within bacterial communities can be challenging, the main advantage of this technique is that it produces a population of differentiated swarmer cells that exist in a single cell layer readily available for single cell microscopy analysis.The implications of this technique extend toward a better understanding of how bacteria regulate changes in the cell cycle, cell morphology, and intracellular organization during cellular differentiation. Demonstration of this method is critical as reliable induction of cellular differentiation and the formation of cells available for single cell analysis are difficult to achieve. To prepare the agar solution for making swarming plates, suspend four grams of heart infusion, or H.I.agar powder, and 100 milliliters of double distilled water.Carefully boil the H.I.agar solution in a microwave oven, shaking the bottle from time to time until the powder is dissolved. Autoclave the agar solution at 121 degrees Celsius for 20 minutes, cool down the agar solution to 60 t0 65 degrees Celsius with constant stirring. The next step involves handling 2, 2'Bipyridyl, which is highly toxic, so gloves and safety goggles must be worn.Add 2, 2'Bipyridyl to a final concentration of 50 micromolar. Add calcium chloride to a final concentration of four millamolar. Inoculate a small amount of V.parahaemolyticus cells from the edge of a single colony into 5 millilters of LB broth.Incubate the culture at 37 degrees Celsius in a shaker incubator until it reaches an optical density at 600 nanometers of 0.8. This usually takes around two to three hours. While the cell culture is growing, pipette 30 to 35 milliliters of the final H.I.agar solution into a round 150 millimeter petri dish, and let the agar solidify.Right before spotting the cell culture, dry the agar plate upright with an open lid at 37 degrees Celsius for at least 10 minutes, or until any liquid residues have disappeared, but no longer. Spot one microliter of cell culture at the center of the H.I.agar swarming plate and let the spot dry. Seal the plate with clear plastic tape, taking care to avoid the formation of air bubbles and folds to the tape.Incubate the plate at 24 degrees Celsius overnight. This will result in the formation of a V.Parahaemolyticus swarm colony with swarm flares extending from the center of the colony, as shown in this time lapse video clip. The first step in this procedure is to prepare agarose slides for microscopy.Add one gram of agarose to 100 milliliters of a 20%PBS and a 10%LB broth solution. Carefully boil the solution in the microwave to dissolve the agarose, and then let it cool down to 65 to 70 degrees Celsius while mixing with a magnetic stir bar. Place a clean microscope slide on to a perfectly level section of a working bench.Fix the slide to the bench by covering both ends of the slide with two layers of general purpose laboratory labeling tape. The distance between the two pieces of tape should be around three centimeters. Pipette about 250 microliters of the previously prepared agarose solution onto the non-covered glass surface.Place a second microscope slide in the same orientation as the bottom slide on top and slightly press down so that the residual agarose can flow out to the sides. Let the agarose solidify for one to two minutes. Use a scalpel to cut off any residual agarose that is attached to the sides of the microscope slide.When the agarose has solidified, slowly pull the top glass slide off of the bottom one in a horizontal movement. Slowly remove the tape from the ends of the microscope slide. The agarose slide should be used as soon as possible, as it will slowly begin to dry out.Immediately before the transfer of swarmer cells to the microscope slide, Use a scalpel to cut the plastic tape off the previously prepared swarmer plate. During the excision of the swarming agar, it is important to direct the cut from the outside to the inside of the swarming colony to avoid contaminating the swarming flares with cells that originate from the middle of the colony. Use a fresh scalpel to cut out a three millimeter by 10 millimeter piece of swarming agar from the most outer edge of a swarming colony, making sure to direct the cuts from the outside of the swarming colony edge to the inside of the colony.Transfer the piece of swarming agar to the microscope agarose slide with the cells facing the agarose pad to imprint the swarming cells onto the agarose pad. After waiting for about 30 seconds, remove the agar piece carefully from the agarose pad. Place a glass coverslip on the agarose pad where the cells were imprinted.The cells are now ready for fluorescence microscopy. Stereo-microscopy imaging of a V.parahaemolyticus swarmer colony in increasing magnification shows swarm flares extending from the periphery of the swarm colony. Cells in the flares are grouped in mono or bilayers only, while cells in the middle of the swarm colony are stacked in multiple layers.DIC microscopy imaging revealed the flares only consist of a fully differentiated population of swarmer cells, while cells from the middle of the swarm colony are significantly shorter, and likely represent a mixture of swimmer and swarmer cells. As proof of principle, DIC and fluorescence microscopy imaging was performed on wild-type V.Parahaemolyticus swimmer and swarmer cells, ectopically expressing an essential chemotaxis protein, YFP-CheW. In swimmer cells, YFP-CheW localizes in a unipolar and bipolar manner, but in swarmer cells, YFP-CheW is always bipolarly localized and also forms clusters positioned randomly along the cell length.Changes in the localization pattern of YFP-CheW during swimmer to swarmer cell differentiation, were also shown by displays of foci distribution and demographic analyses of fluorescence intensity profiles. After its development, this technique paved the way for researchers in the field of microbial cell biology to explore how the bacteria Vibrio parahaemolyticus regulates its internal organization during its distinct differentiated states of swimmer and swarmer cells. After watching this video, you should have a good understanding of how to induce cellular differentiation of the bacteria Vibrio parahaemolyticus and how to prepare a population of differentiated swarmer cells for single cell analysis by fluorescence microscopy.Don't forget that Vibrio parahaemolyticus is a human pathogen and proper procedures and regulations on how to handle BSL 2 pathogens in your region should be applied.

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Immunology and Infection

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 ...

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&amp;#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 ...

Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where immune cells reside and therefore contribute to mucosal immunity in the intestine. A complete understanding of how M cells differentiate in the intestine as well as the molecular mechanisms of antigen uptake by M cells is lacking. This is because M cells are a rare population of cells in the intestine and because in vitro models for M cells are not robust. The discovery of a self-renewing stem cell culture system of the intestine, termed enteroids, has provided new possibilities for culturing M cells. Enteroids are advantageous over standard cultured cell lines because they can be differentiated into several major cell types found in the intestine, including goblet cells, Paneth cells, enteroendocrine cells and enterocytes. The cytokine RANKL is essential in M cell development, and addition of RANKL and TNF-&#945; to culture media promotes a subset of cells from ileal enteroids to differentiate into M cells. The following protocol describes a method for the differentiation of M cells in a transwell epithelial polarized monolayer system of the intestine using human ileal enteroids. This method can be applied to the study of M cell development and function.

This protocol describes how to induce the differentiation of M cells in human stem cell-derived ileal monolayers and methods to assess their development.

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where ...

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be ...

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 million IBD patients. It is generally accepted that a dysregulated immune response to gut bacteria initiates the disease and disrupts the mucosal epithelial barrier. We recently show that gut-homing regulatory T (Treg) cells are a promising therapy for IBD. Accordingly, this article presents a protocol for in vivo augmentation of gut-homing Treg cell induction. In this protocol, dendritic cells are engineered to produce locally high concentrations of two molecules de novo, active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and active vitamin A (retinoic acid or RA). We chose 1,25(OH)2D and RA based on previous findings showing that 1,25(OH)2D can induce the expression of regulatory molecules (e.g., forkhead box P3 and interleukin-10) and that RA can stimulate the expression of gut-homing receptors in T cells. To generate such engineered dendritic cells, we use a lentiviral vector to transduce dendritic cells to overexpress two genes. One gene is the cytochrome P450 family 27 subfamily B member 1 that encodes 25-hydroxyvitamin D 1&#945;-hydroxylase, which physiologically catalyzes the synthesis of 1,25(OH)2D. The other gene is the aldehyde dehydrogenase 1 family member A2 that encodes retinaldehyde dehydrogenase 2, which physiologically catalyzes the synthesis of RA. This protocol can be used for future investigation of gut-homing Treg cells in vivo.

Here we present a protocol for in vivo augmentation of gut-homing regulatory T cell induction. In this protocol, dendritic cells are engineered to locally produce high concentrations of the active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and the active vitamin A (retinoic acid or RA) de novo.

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 ...