Name: application of a mouse ligated peyer&#x2019;s patch intestinal loop assay to evaluate bacterial uptake by m cells
Uploaded: 2011-12-17T14:00:00
Description: Watch this Scientific Journal Video about Application of a Mouse Ligated Peyerâ€™s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells at JoVE.com

The authors would like to thank all EIB members for developing this technique. This research was supported in part by Grant-in-Aid for Scientific Research on Priority Areas "Membrane Traffic" (H.O.), and "Matrix of Infectious Phenomena" (K.H.), Young Scientists (S.F. and K. H.), and Scientific Research (H.O.), and Scientific Research on Innovative Areas "Intracellular Logistics" (H.O.), from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

1. Preparation of bacterial cells
<ol>
	<li>Streak glycerol stocked fluorescent bacteria (such as GFP expressing S. Typhimurium) on LB an agar plate containing 100 &mu;g/ml of ampicillin.</li>
	<li>Culture a single colony from LB agar overnight in 2 ml of new LB medium.</li>
	<li>Add 0.5 ml of bacterial culture to 4.5 ml of new LB medium and incubate until optical density of 1.0 at 600 nm is reached.</li>
	<li>Harvest bacterial cells by centrifugation (3,000 x g, 5 min, 4&deg;C).</li>
	<li>Discard the su...

<ol>
	<li>Fagarasan, S., Honjo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+IgA+synthesis:+regulation+of+front-line+body+defences.">Intestinal IgA synthesis: regulation of front-line body defences.</a> Nat. Rev. Immunol. 3, 63-72 (2003).</li><li>Hapfelmeier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+microbial+colonization+of+germ-free+mice+reveals+the+dynamics+of+IgA+immune+responses.">Reversible microbial colonization of germ-free mice reveals the dynamics of IgA immune responses.</a> Science. 328, 1705-1709 (2010).</li><li>Owen, R. L., Jones, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell+specialization+within+human+Peyer's+patches:+an+ultrastructural+study+of+intestinal+lymphoid+follicles.">Epithelial cell specialization within human Peyer's patches: an ultrastructural study of intestinal lymphoid follicles.</a> Gastroenterology. 66, 189-203 (1974).</li><li>Neutra, M. R., Mantis, N. J., Kraehenbuhl, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collaboration+of+epithelial+cells+with+organized+mucosal+lymphoid+tissues.">Collaboration of epithelial cells with organized mucosal lymphoid tissues.</a> Nat. Immunol. 2, 1004-1009 (2001).</li><li>Hase, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+through+glycoprotein+2+of+FimH(+)+bacteria+by+M+cells+initiates+mucosal+immune+response.">Uptake through glycoprotein 2 of FimH(+) bacteria by M cells initiates mucosal immune response.</a> Nature. 462, 226-230 (2009).</li><li>Punyashthiti, K., Finkelstein, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteropathogenicity+of+Escherichia+coli.+I.+Evaluation+of+mouse+intestinal+loops.">Enteropathogenicity of Escherichia coli. I. Evaluation of mouse intestinal loops.</a> Infect. Immun. 4, 473-478 (1971).</li><li>Owen, R. L., Pierce, N. F., Apple, R. T., Cray, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M+cell+transport+of+Vibrio+cholerae+from+the+intestinal+lumen+into+Peyer's+patches:+a+mechanism+for+antigen+sampling+and+for+microbial+transepithelial+migration.">M cell transport of Vibrio cholerae from the intestinal lumen into Peyer's patches: a mechanism for antigen sampling and for microbial transepithelial migration.</a> J. Infect. Dis. 153, 1108-1118 (1986).</li><li>Owen, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M+cells--entryways+of+opportunity+for+enteropathogens.">M cells--entryways of opportunity for enteropathogens.</a> J. Exp. Med. 180, 7-9 (1994).</li><li>Clark, M. A., Hirst, B. H., Jepson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M-cell+surface+beta1+integrin+expression+and+invasin-mediated+targeting+of+Yersinia+pseudotuberculosis+to+mouse+Peyer's+patch+M+cells.">M-cell surface beta1 integrin expression and invasin-mediated targeting of Yersinia pseudotuberculosis to mouse Peyer's patch M cells.</a> Infect. Immun. 66, 1237-1243 (1998).</li><li>Jensen, V. B., Harty, J. T., Jones, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+the+invasive+pathogens+Salmonella+typhimurium,+Listeria+monocytogenes,+and+Shigella+flexneri+with+M+cells+and+murine+Peyer's+patches.">Interactions of the invasive pathogens Salmonella typhimurium, Listeria monocytogenes, and Shigella flexneri with M cells and murine Peyer's patches.</a> Infect. Immun. 66, 3758-3766 (1998).</li><li>Nagai, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Peyer's+patches+in+the+induction+of+Helicobacter+pylori-induced+gastritis.">Role of Peyer's patches in the induction of Helicobacter pylori-induced gastritis.</a> Proc. Natl. Acad. Sci. U. S. A. 104, 8971-8976 (2007).</li></ol>

The incubation time of ligated Peyer's patch with bacterial suspension is usually for 1 hr to observe the bacterial incorporation into M cells. In case of 4 hrs incubation, the bacteria are often detected in the T cell zone of Peyer's patches. As the vaporized isoflurane inhalant anesthesia could keep mice stable, the incubation time of ligated Peyer's patch with bacterial suspension is extendable to observe the bacteria which move into T-cell zone in Peyer's patch. The important point of this experiment is to take care ...

<table border="1">
	<tbody>
		<tr>
			<th>
				Name of the reagent</th>
			<th>
				Company</th>
			<th>
				Catalogue number</th>
			<th>
				Comments (optional)</th>
		</tr>
		<tr>
			<td>LB Agar Ampicillin- 
			100</td>
			<td>SIGMA</td>
			<td>L5667</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cy3 mono-Reactive 
			Dye Pack</td>
			<td>GE Healthcare</td>
			<td>PA23001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 350 
			carboxylic acid, 
			succinimidyl ester</td>
			<td>Invitrogen</td>
			<td>A10168</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Inhalation anesthesia 
			apparatus</td>
			<td>Shinano Seisakusho</td>
			<td>SN-487</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fixation and 
			Permeabilization 
			Solution</td>
			<td>BD</td>
			<td>554722</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti mouse 
			Glycoprotein 2 
			antibody</td>
			<td>MBL</td>
			<td>D278-3</td>
			<td>200-fold dilution (5&mu;g/ml)</td>
		</tr>
		<tr>
			<td>Goat Anti-Rat IgG, 
			F(ab')2 fragment 
			specific</td>
			<td>Jackson 
			ImmunoResearch</td>
			<td>112-505-006</td>
			<td>200-fold dilution (20&mu;g/ml)</td>
		</tr>
		<tr>
			<td>Alexa Fluor 633 
			Phalloidin</td>
			<td>Molecular Probes</td>
			<td>A22284</td>
			<td>50-fold dilution</td>
		</tr>
		<tr>
			<td>Confocal laser 
			scanning microscope</td>
			<td>Leica Microsystems</td>
			<td>DMIRE2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DeltaVision 
			Restoration 
			deconvolution 
			microscope</td>
			<td>Applied Precision</td>
			<td>DeltaVision Core</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

application of a mouse ligated peyer&#x2019;s patch intestinal loop assay to evaluate bacterial uptake by m cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Watch this Scientific Journal Video about Application of a Mouse Ligated Peyerâ€™s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells at JoVE.com

Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Research

JoVE Journal

Immunology and Infection

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

Cell Membrane Repair Assay Using a Two-photon Laser Microscope

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can ...