This study was supported by Vanderbilt Clinical and Translational Science Award Grant UL1TR002243 (to A.K.) from the National Center for Advancing Translational Sciences; American Heart Association Grant POST903428 (to J.A.I.); and National Heart, Lung, and Blood Institute Grants K01HL13049, R03HL155041, R01HL144941 (to A.K.), and NIH grant 1P01HL116263 (to V.K.). Figure 1 was created using Biorender.

Vanderbilt University&#39;s Institutional Animal Care and Use Committee approved all procedures described in this manuscript. C57B1/6 male mice at 3 months of age, purchased from The Jackson Laboratory, were housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals.
1. Collection, storage, and processing of human fecal samples
<ol>
	<li>Collect a stool sample, using a sterile container if the subject is in the clinic. Refrigerate the stool samples at 4 &#176;C within 36 h of collection until ready for processing. Alternatively, collect stool samples using a commercially available tool for easy and extended DNA stability at ambient temperature, especially for at-home use.</li>
	<li>Sanitize a biosafety-level fume hood with a 10% bleach solution, or other Environmental Protection Agency-approved disinfectant.</li>
	<li>Remove the stool from cool storage and bring into the fume hood; use a disposable spatula to make ~1 g aliquots and store in a -80 &#176;C freezer until completely ready for processing.</li>
	<li>Discard all disposable items in biohazard trash. Disinfect all surfaces (hood and any surfaces touched by the processor) and items being removed from the hood.</li>
</ol>
2. Aseptic collection of mouse fecal samples
NOTE: Use aseptic techniques, including sterilized instruments.
<ol>
	<li>Euthanize the mouse by CO2 asphyxiation. Spray the chest and sides of the mouse with 70% ethanol and carefully open the skin and peritoneal cavity to expose the gastrointestinal tract.</li>
	<li>Isolate the cecum and use sterile surgical scissors to cut it in half. Briefly, expose the cecum and cut 0.5 cm proximally from the ileum and 0.5 cm distally at its junction with the colon. Transfer the isolated cecum onto a sterile Petri dish.</li>
	<li>Use a sterile spatula to transfer cecal content into sterile tubes, and store aliquots in a -80 &#176;C freezer19. 
	&#8203;NOTE: Since the majority of bacteria in the gut are anaerobes, exposure to oxygen may damage or kill the organisms during the isolation procedure in a room atmosphere. Thus, fecal samples should be isolated in anaerobic chambers to maintain the viability of the bacteria.</li>
</ol>
3. Fecal matter transplantation
<ol>
	<li>Resuspend fresh or previously frozen fecal pellets in sterile saline in a 1:20 (w:v) proportion and vortex until homogenized.</li>
	<li>Pass the homogenate through a 30 &#181;m pore nylon filter to remove large particulate matter. Centrifuge at 79 &#215; g for 5 min and collect the supernatant to use for transplantation.</li>
	<li>Oral gavage 100 &#181;L of the slurry per germ-free recipient mouse for 3 consecutive days, followed by gavage every 3 days for 2 weeks. Use conventional mice to study mechanisms of the gut microbiota if they have been first treated with antibiotics to eliminate the recipient&#39;s own endemic microbiota. For example, administer ceftriaxone (400 mg/kg) daily to the recipient mice for 5 consecutive days by oral gavage before gavage of the fecal slurry. 
	NOTE: Studies suggest that a minimum of 2 weeks of this treatment is necessary to elicit cardiovascular changes, including blood pressure20.</li>
	<li>Ensure that germ-free recipient mice are single-housed in gnotobiotic film isolators and fed with sterile food and water.</li>
</ol>
4. Systolic blood pressure measurements
NOTE: Gnotobiotic mice that received FMT from conventionally housed 3-month-old C57Bl/6 mice were implanted with osmotic minipumps (Alzet, model 2002) for infusion of low-dose angiotensin II (140 ng/kg/min) for 2 weeks. Blood pressure was monitored weekly via tail cuff. The protocol for implanting osmotic minipumps has been previously reported21. Tail-cuff was performed as briefly summarized below. A noninvasive method of measuring blood pressure, such as tail cuff, is suitable for FMT studies in gnotobiotic mice. The detailed steps on how to perform tail cuff have been described previously22.
<ol>
	<li>Briefly, retrieve the mice from the gnotobiotic isolators and preheat the tail-cuff machine platform and mouse holder.</li>
	<li>Place the conscious mice in restraints on the heated platform and collect at least three rounds of systolic pressure measurements using tail cuff plethysmography. Perform the following steps below on 3 consecutive days prior to the proper measurement days, to train mice to being restrained in order to reduce stress.
	<ol>
		<li>Gently place the mouse in the preheated holder and leave the tail outside. Carefully tape down the top without pinching it, so as not to stress the mouse.</li>
		<li>Allow the mouse to rest in the holder; place on the platform for 3-5 min covered by a sheet to acclimate.</li>
	</ol>
	</li>
	<li>Average the measurements from all the rounds for an average systolic pressure for each animal.</li>
</ol>
5. Assessment of FMT to cardiovascular changes
<ol>
	<li>Following blood pressure measurement, euthanize the mice and aseptically collect cecal contents, as described in section 2 .</li>
	<li>Harvest the intestines and other tissues, including the heart, aorta, liver, mesenteric arteries, and kidneys, to examine the role of the gut microbiota in cardiometabolic health. To harvest tissues, locate the tissue in the mouse and use scissors to excise them.</li>
	<li>Run a metagenomic sequencing analysis on fecal samples/cecal contents collected from the donor and recipient mice to confirm engraftment of the gut microbiota after FMT23. The first proof of successful colonization of microbiota is confirming that the donor and recipient&#39;s microbiota are similar.</li>
	<li>Use the Swiss-roll technique (see section 6) on the harvested intestinal tissue, coupled with immunostaining and histology to examine morphological and cellular expression changes24.</li>
</ol>
6. Making gut intestine Swiss-rolls
<ol>
	<li>Day 1
	<ol>
		<li>In a properly euthanized mouse sprayed with 70% ethanol, dissect the mouse gut from the anal side (fixed in retroperitoneum) to the stomach side. Place the entirety of the isolated gastrointestinal tract in a Petri dish containing phosphate-buffered saline (PBS). Gently hold the proximal end from the stomach end and remove the surrounding fat and connective tissue by hand.</li>
		<li>Isolate the small intestine (cephalad from the appendix) and make a Z-type zigzag with each length. Then, cut to obtain the duodenum, jejunum, and ileum successively, as previously described25. Isolate the colon by cutting the section of the intestine below the cecum.</li>
		<li>Cut the duodenum, jejunum, ileum, and colon.</li>
		<li>Flush and wash the gut inside using PBS with a syringe and a needle with a ball tip, so as not to tear the intestine.</li>
		<li>Put the gut on filter paper. Label the paper with the name of the section (e.g., duodenum) and then &#39;P&#39; at the right top corner for proximal or &#39;D&#39; for distal at the right bottom corner.</li>
		<li>Cut the gut longitudinally with ball-tip scissors. Open the gut on the filter paper. Wash with more PBS as needed.</li>
		<li>Sandwich the gut between two filter papers. Staple the filter papers at four points/corners near the gut.</li>
		<li>Soak in 10% formalin neutral buffer solution (4.0 g of sodium phosphate, monobasic, 6.5 g of sodium phosphate, dibasic, 100 mL of 37% formaldehyde, 900 mL of distilled water). Shake using a platform rocker at 5 rpm at room temperature overnight.</li>
	</ol>
	</li>
	<li>Day 2
	<ol>
		<li>Prepare 2% agarose in distilled water and heat with a stir bar in a beaker covered with aluminum foil.</li>
		<li>Retrieve the tissues; strip the upper filter paper. Roll the gut from the proximal side so that the proximal side goes inside first, and roll inward so that the lumen is inside on the slide as well. Pin with a 30 G needle or two, as needed.</li>
		<li>Aspirate 1 mL of agarose using disposable graduate transfer pipettes, and pour the agarose on a rolled gut section on a flat surface while avoiding air bubbles in the tissues.</li>
		<li>Allow the agarose to cool and solidify. Use a razor blade to trim the extra agarose around the tissue section.</li>
		<li>Put the gut sections in tissue processing/embedding cassettes (bigger than the regular ones to accommodate the increased height due to agarose). Soak in 70% ethanol at 4 &#176;C.</li>
		<li>Prepare paraffin-embedded tissue slides and proceed to immunostaining, as outlined below.</li>
	</ol>
	</li>
</ol>
7. Immunostaining of the gut intestinal tract
<ol>
	<li>Deparaffinization
	<ol>
		<li>Pass through the following baths with slides in a rack: xylene for 3 min, fresh xylene again for 3 min, xylene with 100% ethanol (1:1) for 3 min, 95% ethanol for 3 min, 70% ethanol for 3 min, and 50% ethanol for 3 min.</li>
		<li>Rinse gently with cold running tap water. Store in a bath of tap water.</li>
	</ol>
	</li>
	<li>Antigen retrieval
	<ol>
		<li>Following deparaffinization, boil the slides in a rack in a bath of antigen retrieval buffer (0.01M trisodium citrate dihydrate at pH 6 and 0.05% Tween-20) at 100 &#176;C for 20 min.</li>
		<li>Run under cold tap water.</li>
	</ol>
	</li>
	<li>Staining
	<ol>
		<li>Remove the slides from the bath and place the tissue face up in a slide box with wet laboratory wipes/paper towels in the bottom. Draw an outline around the tissue with a hydrophobic marker pen.</li>
		<li>Drop Tris-buffered saline (TBS) + 0.025% Triton X-100 onto the tissue and incubate for 5 min. Repeat this step.</li>
		<li>Block with TBS + 10% fetal bovine serum (FBS) + 1% bovine serum albumin (BSA) for 2 h at room temperature. Turn the slides on their side and remove the blocking buffer on a laboratory wipe.</li>
		<li>Add primary antibody solution and incubate at 4 &#176;C for at least 2 h or overnight. Wash gently with TBS + 0.025% Triton X-100 by gently pipetting ~200 &#181;L over the section.</li>
		<li>Add secondary antibody solution and incubate for 1 h at room temperature. Rinse the slides 3 x 5 min with TBS by rinsing with a pipette, as in step 7.3.4.</li>
		<li>Mount with mounting medium and a coverslip.</li>
	</ol>
	</li>
</ol>

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J., 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=Broad+scope+method+for+creating+humanized+animal+models+for+animal+health+and+disease+research+through+antibiotic+treatment+and+human+fecal+transfer.">Broad scope method for creating humanized animal models for animal health and disease research through antibiotic treatment and human fecal transfer.</a> Gut Microbes. 5 (2), 183-191 (2014).</li><li>Wilde, 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=Tail-cuff+technique+and+its+influence+on+central+blood+pressure+in+the+mouse.">Tail-cuff technique and its influence on central blood pressure in the mouse.</a> Journal of the American Heart Association. 6 (6), 005204 (2017).</li><li>Liu, X., 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=High-fiber+diet+mitigates+maternal+obesity-induced+cognitive+and+social+dysfunction+in+the+offspring+via+gut-brain+axis.">High-fiber diet mitigates maternal obesity-induced cognitive and social dysfunction in the offspring via gut-brain axis.</a> Cell Metabolism. 33 (5), 923-938 (2021).</li></ol>

No conflicts of interest, financial or otherwise, are declared by the authors.

A valuable approach to studying the causal role of gut microbiota in cardiovascular and metabolic disease is to transfer the total microbiota or select species of interest into germ-free mice. Here, we describe protocols to collect fecal samples from humans and conventionally housed mice into germ-free mice to study the role of gut microbiota in hypertensive disorders.
In mice, we use aseptically collected cecal contents processed in an aerobic chamber, and in humans, collect feces. FMT can be...

Cardiometabolic disorders, including heart disease and stroke, are the leading global causes of death1. Physical inactivity, poor nutrition, advancing age, and genetics modulate the pathophysiology of these disorders. Accumulating evidence supports the concept that gut microbiota affect cardiovascular and metabolic disorders, including type 2 diabetes2, obesity3, and hypertension4, which may hold a key to the development of new therapeutic approaches for these diseases.
The exact mechanisms by which the microbiota cause diseases are still unknown, and current studies are highly variable, in part due to methodological differences. Fecal microbiota transplantation (FMT) is a valuable approach to delineating a direct role of the total microbiota or isolated species in disease pathophysiology. FMT is widely used in animal studies to induce or suppress a phenotype. For example, caloric intake and glucose metabolism can be modulated by transferring fecal matter from a sick donor to a healthy recipient5,6. In humans, FMT has been shown to be a safe treatment option for patients with recurrent Clostridium difficile infection7. Evidence supporting its use in cardiovascular disease management is emerging; for instance, FMT from lean to metabolic syndrome patients improves insulin sensitivity8. Gut dysbiosis is also associated with high blood pressure in both human and rodent studies9,10,11. FMT from mice fed a high salt diet into germ-free mice predisposes the recipients to inflammation and hypertension12.
Despite the high rate of FMT success in rodents, translational challenges remain. Clinical trials using FMT to treat obesity and metabolic syndrome indicate minimal to no effects on these disorders13,14,15. Thus, more studies are needed to identify additional therapeutic avenues targeting the gut microbiota for the treatment of cardiometabolic disorders. Most of the available evidence on the gut microbiota and cardiovascular disease is associative. The described protocol discusses how to utilize a combination of FMT and the Swiss-rolling technique to show both an association between disease and gut microbiota and directly assess the integrity of all parts of the gut intestine16,17,18.
The overall goal of this method is to provide guidance for studying the effects of the gut microbiome in experimental cardiovascular disease. This protocol provides more details and key considerations in the experimental design to promote physiological translation and increase the rigor and reproducibility of the findings.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>Alexa Fluor 488 Tyamide SuperBoost</td>
			<td>ThermoFisher</td>
			<td>B40932</td>
			<td></td>
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		<tr>
			<td>Anaerobic chamber</td>
			<td>COY</td>
			<td>7150220</td>
			<td></td>
		</tr>
		<tr>
			<td>Apolipoprotein AI</td>
			<td>Novus Biologicals</td>
			<td>NBP2-52979</td>
			<td></td>
		</tr>
		<tr>
			<td>Artery Scissors - Ball Tip</td>
			<td>Fine Science Tools</td>
			<td>14086-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach solution</td>
			<td>Fisher Scientific</td>
			<td>14-412-53</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher Scientific</td>
			<td>B14</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3 antibody</td>
			<td>ThermoFisher</td>
			<td>&#160;14-0032-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD68 monoclonal antibody</td>
			<td>ThermoFisher</td>
			<td>14-0681-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Fisher Scientific</td>
			<td>75-004-221</td>
			<td></td>
		</tr>
		<tr>
			<td>CODA high throughput monitor</td>
			<td>Kent Scientic Corporation</td>
			<td>CODA-HT8</td>
			<td></td>
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			<td>Cryogenic vials</td>
			<td>Fisher Scientific</td>
			<td>10-500-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable graduate transfer pipettes</td>
			<td>Fisher Scientific</td>
			<td>137119AM</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable syringes</td>
			<td>Fisher Scientific</td>
			<td>14-823-2A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>AA33361M1</td>
			<td></td>
		</tr>
		<tr>
			<td>Feeding Needle</td>
			<td>Fine Science Tools</td>
			<td>18061-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter (30 &#181;m)</td>
			<td>Fisher Scientific</td>
			<td>NC0922459</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper sheet</td>
			<td>Fisher Scientific</td>
			<td>09-802</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin (10%)</td>
			<td>Fisher Scientific</td>
			<td>23-730-581</td>
			<td></td>
		</tr>
		<tr>
			<td>High salt diet</td>
			<td>Teklad</td>
			<td>TD.03142</td>
			<td></td>
		</tr>
		<tr>
			<td>OMNIgene.GUT</td>
			<td>DNAgenotek</td>
			<td>OM-200+ACP102</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmotic mini-pumps</td>
			<td>Alzet</td>
			<td>&#160;MODEL 2002</td>
			<td></td>
		</tr>
		<tr>
			<td>PAP Pen</td>
			<td>Millipore Sigma</td>
			<td>Z377821-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Fisher Scientific</td>
			<td>AS4050</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>Fisher Scientific</td>
			<td>21-236-18C</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Fisher Scientific</td>
			<td>14-388-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Serile Phosphate-buffered saline</td>
			<td>Fisher Scientific</td>
			<td>AAJ61196AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Smart spatula</td>
			<td>Fisher Scientific</td>
			<td>NC0133733</td>
			<td></td>
		</tr>
		<tr>
			<td>Stool collection device</td>
			<td>Fisher Scientific</td>
			<td>50-203-7255</td>
			<td></td>
		</tr>
		<tr>
			<td>TBS Buffer</td>
			<td>Fisher Scientific</td>
			<td>R017R.0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Millipore Sigma</td>
			<td> 
			9036-19-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Varimix platform rocker</td>
			<td>Fisher Scientific</td>
			<td>09047113Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Fisher Scientific</td>
			<td>02-215-41</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Fisher Scientific</td>
			<td>1330-20-7, 100-41-4</td>
			<td></td>
		</tr>
	</tbody>
</table>

murine fecal isolation and microbiota transplantation

Gut microbiota dysbiosis plays a role in the pathophysiology of cardiovascular and metabolic disorders, but the mechanisms are not well understood. Fecal microbiota transplantation (FMT) is a valuable approach to delineating a direct role of the total microbiota or isolated species in disease pathophysiology. It is a safe treatment option for patients with recurrent Clostridium difficile infection. Preclinical studies demonstrate that manipulating the gut microbiota is a useful tool to study the mechanistic link between dysbiosis and disease. Fecal microbiota transplantation may help elucidate novel gut microbiota-targeted therapeutics for the management and treatment of cardiometabolic disease. Despite a high success rate in rodents, there remains translational changes associated with the transplantation. The goal here is to provide guidance in studying the effects of gut microbiome in experimental cardiovascular disease. In this study, a detailed protocol for the collection, handling, processing, and transplantation of fecal microbiota in murine studies is described. The collection and processing steps are described for both human and rodent donors. Lastly, we describe using a combination of the Swiss-rolling and immunostaining techniques to assess gut-specific morphology and integrity changes in cardiovascular disease and related gut microbiota mechanisms.

The steps described above are summarized in Figure 1. Mouse cecal contents or human feces are resuspended in sterile saline to prepare a slurry to give to germ-free mice (100 &#181;L) by gavage, first for 3 consecutive days, then once every 3 days. At the end of the protocol, blood pressure is measured by the tail-cuff method, mice are euthanized, and tissues are harvested for assessment of changes in the gut microbiota and cardiovascular and metabolic changes.
A ...

Watch this Scientific Journal Video about Murine Fecal Isolation and Microbiota Transplantation at JoVE.com

Murine Fecal Isolation and Microbiota Transplantation

The goal here is to outline a protocol to investigate the mechanisms of dysbiosis in cardiovascular disease. This paper discusses how to aseptically collect and transplant murine fecal samples, isolate intestines, and use the "Swiss-roll" method, followed by immunostaining techniques to interrogate changes in the gastrointestinal tract.

Gut microbiota dysbiosis plays a role in the pathophysiology of cardiovascular and metabolic disorders, but the mechanisms are not well understood. Fecal ...

murine-fecal-isolation-and-microbiota-transplantation

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Immunology and Infection

3.5K Views.  Vanderbilt University. The goal here is to outline a protocol to investigate the mechanisms of dysbiosis in cardiovascular disease. This paper discusses how to aseptically collect and transplant murine fecal samples, isolate intestines, and use the "Swiss-roll" method, followed by immunostaining techniques to interrogate changes in the gastrointestinal tract. 

Video: Murine Fecal Isolation and Microbiota Transplantation

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory bowel disease, asthma and arthritis. Thus, understanding the mechanisms underlying microbiota-immune system crosstalk is of crucial importance. Antibiotic administration, while aiding pathogen clearance, also induces drastic changes in the size and composition of intestinal bacterial communities which can have an impact on human health. Antibiotic treatment in mice recapitulates the impact and long-term changes in human microbiota from antibiotic treated patients, and enables investigation of the mechanistic links between changes in microbial communities and immune cell function. While several methods for antibiotic treatment of mice have been described, some of them induce severe dehydration and weight-loss complicating the interpretation of the data. Here, we provide two protocols for oral antibiotic administration which can be used for long-term treatment of mice without inducing major weight-loss. These protocols make use of a combination of antibiotics that target both Gram-positive and Gram-negative bacteria and can be provided either ad libitum in the drinking water or by oral gavage. Moreover, we describe a method for the quantification of microbial density in fecal samples by qPCR which can be used to validate the efficacy of the antibiotic treatment. The combination of these approaches provides a reliable methodology for the manipulation of the intestinal microbiota and the study of the effects of antibiotic treatment in mice.

Here we provide detailed protocols for the oral administration of antibiotics to mice, collection of fecal samples, DNA extraction and quantification of fecal bacteria by qPCR.

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory ...

Using Robotic Systems to Process and Embed Colonic Murine Samples for Histological Analyses

The understanding of human diseases has been greatly expanded thanks to the study of animal models. Nonetheless, histopathological evaluation of experimental models needs to be as rigorous as that applied for human samples. Indeed, drawing reliable and accurate conclusions is critically influenced by the quality of tissue section preparation. Here, we describe a protocol for histopathological analysis of murine tissues that implements several automated steps during the procedure, from the initial preparation to the paraffin embedding of the murine samples. The reduction of methodological variables through rigorous protocol standardization from automated procedures contributes to increased overall reliability of murine pathological analysis. Specifically, this protocol describes the utilization of automated processing and embedding robotic systems, routinely used for the tissue processing and paraffin embedding of human samples, to process murine specimens of intestinal inflammation. We conclude that the reliability of histopathological examination of murine tissues is significantly increased upon introduction of standardized and automated techniques.

Lack of standardization for murine tissue processing reduces the quality of murine histopathological analysis as compared to human specimens. Here, we present a protocol to perform histopathological examination of murine inflamed and uninflamed colonic tissues to show the feasibility of robotic systems routinely used for processing and embedding&#160;human samples.

The understanding of human diseases has been greatly expanded thanks to the study of animal models. Nonetheless, histopathological evaluation of ...

An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are needed to decipher the utilization of microbiome modulators for human health benefits. However, the large-scale screening and optimization of modulators to validate microbiome modulation and predict related health benefits may be practically difficult due to the need for large number of animals and/or human subjects. To this end, in vitro or ex vivo models can facilitate preliminary screening of microbiome modulators. Herein, it is optimized and demonstrated an ex vivo fecal microbiota culture system that can be used for examining the effects of various interventions of gut microbiome modulators including probiotics, prebiotics and other food ingredients, aside from nutraceuticals and drugs, on the diversity and composition of the human gut microbiota. Inulin, one of the most widely studied prebiotic compounds and microbiome modulators, is used as an example here to examine its effect on the healthy fecal microbiota composition and its metabolic activities, such as fecal pH and the fecal levels of organic acids including lactate and short-chain fatty acids (SCFAs). The protocol may be useful for studies aimed at estimating the effects of different interventions of modulators on fecal microbiota profiles and at predicting their health impacts.

This protocol describes an in vitro batch-culture fermentation system of human fecal microbiota, using inulin (a well-known prebiotic and one of the most widely studied microbiota modulators) to demonstrate the use of this system in estimating effects of specific interventions on fecal microbiota composition and metabolic activities.

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are ...

Renal Subcapsular Transplantation of 2'-Deoxyguanosine-Treated Murine Embryonic Thymus in Nude Mice

The thymus is an important central immune organ, which plays an essential role in the development and differentiation of T cells. Thymus transplantation is an important method for investigating thymic epithelial cell function and T cells maturation in vivo. Here we will describe the experimental methods used within our laboratory to transplant 2&#8217;-deoxyguanosine (to deplete donor&#8217;s lymphocytes) treated embryonic thymus into the renal capsule of an athymic nude mouse. This method is both simple and efficient and does not require special skills or devices. The results obtained via this simple method showed that transplanted thymus can effectively support the recipient&#8217;s T cells production. Additionally, several key points with regards to the protocol will be further elucidated.

We provide a simple and efficient method to transplant 2&#8217;-deoxyguanosine treated E18.5 thymus into the renal capsule of a nude mouse. This method should aide in the study of both thymic epithelial cells function and T cells maturation.

The thymus is an important central immune organ, which plays an essential role in the development and differentiation of T cells. Thymus transplantation ...