Name: isolation of salivary epithelial cells from human salivary glands for in vitro growth as salispheres or monolayers
Uploaded: 2019-07-15T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers at JoVE.com

We would like to thank James P. Bridges for providing significant guidance on the methodologies involved for culturing primary cells. Matthew J. Beucler was supported by National Institutes of Health Training Grant T32-ES007250. This work was also supported by National Institutes of Health grants R01-AI121028 and R21-DE026267 awarded to William E. Miller.

These protocols and studies have been reviewed and approved by the Institutional Review Board at the University of Cincinnati (Federal-wide Assurance #00003152, IRB protocol 2016-4183). Salivary tissue is commonly resected in many head and neck surgical procedures and is usually uninvolved by the malignant process. Typically, freshly resected salivary gland tissue is used within 2-4 h after removal from the patient (Figure 1A).
1. Reagent Preparation
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D., 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=Primary+Culture+of+Polarized+Human+Salivary+Epithelial+Cells+for+Use+in+Developing+an+Artificial+Salivary+Gland.">Primary Culture of Polarized Human Salivary Epithelial Cells for Use in Developing an Artificial Salivary Gland.</a> Tissue Engineering. 11, 172-181 (2005).</li><li>Pringle, 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=Isolation+of+Mouse+Salivary+Gland+Stem+Cells.">Isolation of Mouse Salivary Gland Stem Cells.</a> Journal of Visualized Experiments. , (2011).</li><li>Nam, H., 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=Characterization+of+Primary+Epithelial+Cells+Derived+from+Human+Salivary+Gland+Contributing+to+in+vivo+Formation+of+Acini-like+Structures.">Characterization of Primary Epithelial Cells Derived from Human Salivary Gland Contributing to in vivo Formation of Acini-like Structures.</a> Molecules and Cells. 41, 515-522 (2018).</li><li>Vissink, A., Jansma, J., Spijkervet, F. 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Salivary dysfunction represents a concern for the quality of life for those suffering from Sj&#246;gren&#39;s syndrome as well as those undergoing radiation therapy for cancers adjacent to the salivary glands3,4,5,16. One proposed therapy to treat these patients is to grow functional salivary stem cells or organoids in vitro, which can then be inserted into damaged salivary gland to replace aff...

In mammals the salivary glands are organized into 3 pairs of major salivary glands: the parotid, submandibular, and sublingual glands. There also exists a series of minor glands located on the tongue and scattered throughout the oral cavity1. The major glands are responsible for the bulk volume of saliva produced2. In addition to providing antimicrobial protection through secreted factors, the saliva is important in the process of chewing and lubricating food as it passes through the esophagus2. As such, salivary gland dysfunction represents a medical issue where sufferers who are unable to produce enough saliva are more prone to developing maladies such as dental cavities or oral candidiasis3. Additionally, reduced saliva results in greater difficulty eating and digesting food having a significant impact on quality of life.
Salivary gland dysfunction is primarily found in patients suffering from Sj&#246;gren&#39;s syndrome, an autoimmune disorder, or in patients with head and neck cancer who have undergone irradiation therapy3,4,5,6. Damaged secretory acini within the glands as a result of autoimmunity or radiation therapy do not undergo self-renewal, which results in a patient living with irreversible damage6. The study of salivary glands has also garnered the attention or researchers interested in mechanisms of viral pathogenesis. Some pathogens, such as human cytomegalovirus (HCMV), persistently replicate within the salivary glands, which then allows for transmission of nascent virus to a new host via saliva7,8. Thus, there is an unmet need to develop robust and reproducible in vitro models of salivary gland tissue to tackle and understand a diverse array of medical problems.
Several models of the murine salivary gland system have been used as a starting point to understand and characterize the different epithelial cells that form the salivary glands9,10. There exist obvious and major differences between human and murine salivary glands11. Most notably the human parotid gland is larger in relation to the submandibular gland whereas the mouse submandibular and parotid are much similar in size1,2,11. While immortalized human submandibular cell lines exists, there are major concerns that the immortalized cells do not accurately maintain the phenotype of the primary tissue and secondary concerns that the immortalized cells are not actually derived from salivary epithelium itself12. For these reasons there is an interest and a need to study primary salivary tissue from humans.
Here we describe a protocol to isolate primary epithelial cells from human salivary glands for tissue culture. Our protocol is based on the works of Pringle et al. and Tran et al. with modifications made to generally simplify their procedures13,14. First, while Tran et al.&#39;s protocol calls for a long five-hour incubation for enzymatically digesting the salivary tissue, our modified protocol has been successful with as little as one-hour incubation, similar to that in Pringle et al. Second, we use different enzymes for tissue digestion, most notably we don&#39;t use trypsin as is used in the original Tran et al. protocol, but instead use a mixture of dispase and collagenase. We prefer to avoid trypsin in the initial digestion steps as a recent study suggested that initial digestion of the salivary tissue by trypsin results in reduced cell viability, possibly by the loss of a rare stem cell population15. Lastly, we culture the salispheres on the surface of basement membrane matrix (BMM) using a commercially available media originally formulated for the propagation of bronchial epithelial cells. Our protocol can be used to isolate salivary cells from parotid, submandibular, and sublingual glands. These cells express salivary amylase and other salivary specific genes indicating that they maintain a phenotype similar to that of salivary acinar epithelial cells7. We have successfully generated salivary cultures from &#62;50 primary human tissue samples using this methodology. Moreover, the primary salivary cells can easily be cryopreserved for use at a later date.

<table border="0" cellpadding="0" cellspacing="0" style="width:736px;" width="736">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">100 mm culture dishes</td>
			<td style="width:132px;">Thermo Scientific</td>
			<td style="width:123px;">172931</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">15 mL conical tubes</td>
			<td style="width:132px;">Thermo Scientific</td>
			<td style="width:123px;">339651</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">50 mL conical tubes</td>
			<td style="width:132px;">Thermo Scientific</td>
			<td style="width:123px;">339653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Bronchial Epithelial Cell Growth Media</td>
			<td style="width:132px;">Lonza</td>
			<td style="width:123px;">CC-3171</td>
			<td>Add bullet kit as per manufacturer&#39;s instructions. Supplement with 20 mL of charcoal stripped serum.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Cell strainer 70 &#181;m nylon mesh</td>
			<td style="width:132px;">Fisher</td>
			<td style="width:123px;">22-363-548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Charcoal stripped fetal bovine serum</td>
			<td style="width:132px;">Gibco</td>
			<td style="width:123px;">12676-029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Collagenase type III</td>
			<td style="width:132px;">Worthington</td>
			<td style="width:123px;">LS004182</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Cryogenic Tube</td>
			<td style="width:132px;">Fisher</td>
			<td style="width:123px;">5000-0020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Dispase</td>
			<td style="width:132px;">Cell Applications</td>
			<td style="width:123px;">07923</td>
			<td>Dissolve collagenase to make a 0.15% (w/v) stock. Filter sterilize then store at -20 &#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Dissecting scissors</td>
			<td style="width:132px;">Fisher</td>
			<td style="width:123px;">08-940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Dulbecco phosphate buffered saline</td>
			<td style="width:132px;">Corning</td>
			<td style="width:123px;">55-031-PC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">General Chemicals</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">PathClear Basement membrane extract</td>
			<td style="width:132px;">Cultrex</td>
			<td style="width:123px;">3432-005-01</td>
			<td>Thaw at 4 &#176;C at least 24 hr prior to use. Always handle on ice.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Six-well culture dishes</td>
			<td style="width:132px;">Falcon</td>
			<td style="width:123px;">353046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Surgical forceps</td>
			<td style="width:132px;">Fisher</td>
			<td style="width:123px;">22-079-742</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Trypsin-EDTA solution</td>
			<td style="width:132px;">Corning</td>
			<td style="width:123px;">25-052-CI</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of salivary epithelial cells from human salivary glands for in vitro growth as salispheres or monolayers

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to restore function of glands damaged by radiation therapies or due to pathologies associated with Sj&#246;gren's syndrome. A second goal of researchers is to define the mechanisms by which viruses replicate within glandular tissue where they can then gain access to salivary fluids important for horizontal transmission. These goals highlight the need for a robust and accessible in vitro salivary gland model that can be utilized by researchers interested in the above mentioned as well as related research areas. Here we discuss a simple protocol to isolate epithelial cells from human salivary glands and propagate them in vitro. Our protocol can be further optimized to meet the needs of individual studies. Briefly, salivary tissue is mechanically and enzymatically separated to isolate single cells or small clusters of cells. Selection for epithelial cells occurs by plating onto a basement membrane matrix in the presence of media optimized to promote epithelial cell growth. These resulting cultures can be maintained as three-dimensional clusters, termed "salispheres", or grown as a monolayer on treated plastic tissue culture dishes. This protocol results in the outgrowth of a heterogenous population of mainly epithelial cells that can be propagated for 5-8 passages (15-20 population doublings) before undergoing cellular senescence.

Two-three days after plating cells from digested tissue onto BMM, cells will readily form small clusters that will continue to expand in size up to 15-20 cells per cluster (Figure 2A). Cellular debris and detached dead cells are typically seen and should be removed by aspirating and replenishing with fresh media. Cells will continue to proliferate as salispheres for about 3-10 days, or as long as the BMM layer remains intact. Occasionally, due to partial brea...

Watch this Scientific Journal Video about Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers at JoVE.com

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers

We present a method for isolating and cultivating primary human salivary gland-derived epithelial cells. These cells exhibit gene expression patterns consistent with them being of salivary epithelial origin and can be grown as salispheres on basement membrane matrices derived from Engelbreth-Holm-Swarm tumor cells or as monolayers on treated culture dishes.

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to ...

Generating primary cellular models or organoid-like structures is key to answering many contemporary questions in biology. For example, we've been interested for quite some time in the mechanisms used by human viruses to facilitate replication in the salivary gland as it is an important site for horizontal transmission. The development of the salisphere-based system described in this protocol is a crucial advancement in our quest to answer these types of questions.This technique allows us to generate primary cells from a variety of different patient samples. The cellular models are more reminiscent of the in vivo type situation as the cells are not modeled or transformed like many cell types commonly used for in vitro experiments. We've used this protocol to generate similar cells from mouse primary submandibular gland and the protocol would likely be useful for other organs with similar cellular structures.Within two to four hours after removal from the patient, on a culture plate, use autoclaved sterilized dissecting scissors and surgical forceps to mince the salivary gland tissue into fine pieces at the size of one to two millimeters. Add six milliliters of the Dispase/Collagenase solution to the minced tissue. Then incubate at 37 degrees Celsius for approximately 30 minutes to one hour.Disrupt the tissue by pipetting with a five milliliter serological pipette 15 to 20 times. Incubate at 37 degrees Celsius for another 30 minutes to one hour. Repeat the pipetting and incubation step two to three more times or until the tissue resembles a slurry and can easily pass through the pipette opening.To coat wells with BMM, slowly pipette 500 microliters of overnight thawed BMM into each well of a six well plate. Slowly swirl the plate to evenly distribute the BMM over the wells. Then incubate the coated plates at 37 degrees Celsius for at least 15 minutes prior to use to allow the BMM to solidify.First, transfer the Dispase/Collagenase solution containing homogenized tissue to a 15 milliliter conical tube. Wash the wall of the plate once with six milliliters of DPBS to transfer the remaining cells into the conical tube. Then through a 70 micrometer nylon mesh cell strainer, filter the homogenate into a new 50 milliliter conical tube to remove undigested tissue.Centrifuge strained cells for five minutes at 500 times g. Aspirate the supernatant. Then resuspend the cell pellet in 10 milliliters of 1X RBC lysis buffer.Incubate for five minutes at 37 degrees Celsius. Then add 20 to 25 milliliters of DPBS to neutralize the RBC lysis buffer and minimize lysis of salivary cells. Centrifuge for five minutes at 500 times g.White cell pellet indicates complete lysis of all RBC. If the pellet has red color, repeat treatment with RBC lysis buffer. Aspirate the supernatant and resuspend the pellet in one to two milliliters of BEGM and then transfer the resuspended cells onto BMM coated wells.Incubate at 37 degrees Celsius. Once plated on BMM, cells will form into spherical structures or salispheres over a two to three-day period. Cells can be maintained as salispheres for about five to seven days before the BMM begins to degrade allowing the cells to access and adhere to the plastic and grow as a monolayer.First, aspirate the media from the wells and add one milliliter of Dispase/Collagenase solution to each well. Incubate at 37 degrees Celsius for approximately 15 minutes or until BMM has mostly dissolved. Then transfer the cells to a 15 milliliter conical tube and wash wells once with one to three milliliters of DPBS to obtain the remaining cells.Centrifuge for five minutes at 500 times g. Aspirate the supernatant and resuspend the pellet in two milliliters of Trypsin. Incubate again at 37 degrees Celsius for 15 minutes.Next, add in to the tube any complete media containing 10%serum to neutralize the Trypsin. Centrifuge for five minutes at 500 times g. Aspirate the supernatant to remove residual media, Trypsin and serum and then resuspend the cells in DPBS.Centrifuge for five minutes at 500 times g. Aspirate the supernatant and resuspend the pellet in BEGM. To maintain the pellet as spheres, plate the resuspended cells onto solidified BMM coated culture dishes.Culture all cells in a humidified incubator maintained at 37 degrees Celsius with 5%carbon dioxide for five to seven days prior to subculturing. Feed with fresh BEGM every two to three days. To proliferate the cells as a monolayer onto plastic culture dishes, aspirate the BEGM media from the dish and wash once with DBPS to remove residual media.Add two milliliters of Trypsin and incubate at 37 degrees Celsius for 15 minutes or until cells have fully detached. Neutralize Trypsin using four milliliters of any complete media containing 10%serum. Then gently tilt the dish and transfer cells to a 15 milliliter conical tube.Wash the plate once with approximately five milliliters of DPBS to collect the remaining cells. Centrifuge for five minutes at 500 times g. Aspirate the supernatant.To remove residual media, resuspend cells in six to 10 milliliters of DPBS. Centrifuge for five minutes at 500 times g. Aspirate the supernatant and resuspend in BEGM.Then plate the cells onto the treated plastic tissue culture dishes. Culture the cells in a humidified incubator maintained at 37 degrees Celsius with 5%carbon dioxide for five to seven days prior to subculturing. Feed with fresh BEGM every two to three days.In this protocol, after plating cells from the digested tissue onto BMM for two to three days, cells readily formed small clusters that continued to expand in size up to 15 to 20 cells per cluster. Salisphere cells were plated onto cell cultured treated plastic and grown as a monolayer where they exhibit morphology consistent with cells of epithelial origin. Proper aseptic technique and care is needed to avoid bacterial or fungal contamination.No amount of antibiotics or antimycotics will save a culture swarming with microbes. We are answering questions about viral pathogenesis and transmission. But for others, this provides a way to study human salivary cells for any number of applications such as tissue regeneration therapies.For us who study human Cytomegalovirus, this technique has allowed us to begin exploring the molecular mechanisms the virus utilizes to infect and ultimately transmit to new hosts. Nothing used in this protocol is inherently hazardous but caution should always be taken when handling human tissue and any sort of chemical reagents.

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Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

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.

Application of a Mouse Ligated Peyer&amp;#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.

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

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.