This work was supported by National Institutes of Health Grant R01 AI088209 (to K.A.H.). We also thank the University of Minnesota Flow Cytometry Resource.

In this study, adult (6 - 8 weeks) female C57Bl/6 mice were used. Mice were purchased from the National Cancer Institute and maintained under specific pathogen free conditions. The University of Minnesota Institutional Animal Care and Use Committee (IACUC) approved all animal experimentation.
1. Preparation of Instruments and Buffers
<ol>
	<li>Prepare enzyme solution (RPMI1640 medium with 0.05% [w/v] of Liberase TH and 100 U/ml of DNase I), albumin-rich buffer (1X PBS [Ca2+/Mg2+-free] with 0.5% Bovine serum albumin and 2 mM Ethylenediamine Tetraacetic acid [EDTA]), FACS buffer (1X PBS [Ca2+/Mg2+-free] containing 1% Fetal bovine serum [FBS] and 0.02% NaN3), FACS sorting buffer (1X PBS [Ca2+/Mg2+-free] containing 2% FBS), and RP10 medium (RPMI-1640 medium supplied with 10% FBS).</li>
	<li>Prepare 10 panning plates. Prepare 40 ml coating solution (0.01 mg/ml Goat anti-Rat IgG in 0.1 M NaHCO3 Buffer), and add 4 ml of coating solution into each 100 x 15 mm plate, swirl and incubate at 4 &#176;C O/N. Pour off antibody solution and rinse the plate using 5 ml of 1x PBS for 2 times, swirl vigorously between rinses. Store the prepared plates at 4 &#176;C.</li>
</ol>
2. Harvest of Thymus Tissue
<ol>
	<li>Euthanize mouse in a CO2 chamber. Put the animal on its back on a dissection mat, pin the feet on the mat and wet the fur by spraying with 70% ethanol.</li>
	<li>Make a midline incision through the skin, starting from above the urethral opening straight up to the lower jaw with scissors and extend the incision downward to the knees on both sides. The incision looks like an upside down &#8220;Y&#8221;. Pull the loose skin back on the sides, and pin it to the dissection mat.</li>
	<li>Puncture the diaphragm from the xiphoid, cut the ribs on each side up to about the clavicle, then lift up the ribs with forceps and pin it to the dissection mat. The thymus is just under the ribs, and looks like two thin white lobes overlying the heart (Figure 1). Disconnect connective tissue surrounding the thymus, gently pull and remove the pair of thymus lobes with curved serrated forceps. Place thymus lobes into a 6 well plate containing 5 ml RPMI-1640.</li>
</ol>
3. Preparation of Thymic Stromal Cells
<ol>
	<li>Trim any surrounding fat and connective tissue and transfer the thymus lobes to a new well of the 6 well plate containing 5 ml freshly prepare enzyme solution.&#160; Make small incisions in the thymus lobes with fine scissors, and incubate the plate at 37 &#176;C for 20 min.</li>
	<li>Gently pipette digesting thymus tissue up and down 2 - 3 times using 5 ml pipette for tissue dissociation. Collect supernatant fraction in a 50 ml tube containing 10 ml cold albumin-rich buffer to neutralize enzymes.&#160; Keep the collection tube on ice.&#160; Add 2.5 ml enzyme solution to the remaining tissue and incubate the plate at 37 &#176;C for 15 min.</li>
	<li>Perform gentle mechanical agitation with a 3 ml syringe and 18 G needle to break up aggregates.&#160; Pool supernatant into the collection tube.&#160; Add 2.5 ml enzyme solution to the remaining tissue and incubate the plate at 37 &#176;C for 15 min.</li>
	<li>Repeat gentle mechanical agitation with a 3 ml syringe and a 25 G needle, and incubate the plate additional 5 - 10 min.</li>
	<li>After complete digestion, pool the supernatant into the collection tube.&#160; Centrifuge the collection tube at 400 x g, 4 &#176;C for 8 min to collect the cells.&#160; Aspirate the liquid and suspend the pelleted cells in 10 ml albumin-rich buffer and filter the sample through 100 mm mesh.&#160;&#160; Count cells using a hemocytometer.&#160; Then perform flow cytometic analysis (Section 4) or TECs enrichment (Section 5).</li>
</ol>
4. Identification of TECs by Flow Cytometry
<ol>
	<li>Prepare cells to a final concentration of 4 x 106 cells per 100 &#181;l in FACS buffer for staining in a 96 well round-bottom test plate and keep the plate on ice. Add 20 &#181;l Fc block solution (anti-CD16/CD32 antibody at 10 &#181;g/ml in FACS buffer) to the cells and incubate the cells for 10 min on ice. Spin the plate at 400 x g, 4 &#176;C for 3 min. Discard liquid from wells by flicking the plate face down into a sink.</li>
	<li>Suspend the pelleted cells in 100 &#181;l antibody cocktail (anti-CD45, -EpCAM, -MHC class II, and -Ly51 antibodies, and the UEA-1 lectin labeled with different fluorochromes at manufacturer recommended or tested optimal concentrations of each antibody in FACS buffer), and incubate the cells on ice for 20 min in the dark. For compensation, stain 1 x 106 cells with each individual fluorochrome.</li>
	<li>Add 100 &#181;l of FACS buffer to each well and spin the plate at 400 x g, 4 &#176;C for 3 min. Repeat the wash step one time, and then resuspend cells in 100 &#181;l FACS buffer. Transfer the cells to a 12 x 15 mm round-bottom FACS tube filled with 300 &#181;l FACS buffer. Acquire samples on flow cytometer and analyze data using FACS analysis software. TEC gating strategy is shown in Figure 2.</li>
</ol>
5. Enrichment of TECs by Panning
<ol>
	<li>Spin down the thymus cells (prepared in Section 3) at 400 x g, 4 &#176;C for 5 min. Aspirate the liquid and suspend the pelleted cells in FACS sorting buffer at a concentration of 2 x 108 cells per ml in a 15 ml or 50 ml conical tube. Add anti-CD90.2 antibody (Clone 30-H12) (or anti-CD45) at a final concentration of 2.5 &#181;g/ml of cell suspension and incubate cells gently on a rotating mixer at 4 &#176;C for 30 min. Following the incubation, wash with a 5 - 10x volume of RP10 medium and centrifuge the tube at 400 x g, 4 &#176;C for 5 min.</li>
	<li>Aspirate the liquid and resuspend the pelleted cells in RP10 medium at a concentration of 1 x 107 per ml. Add 5 ml cell suspension to each coated panning plate (prepared in Step 1.3), swirl and incubate for 30 min at RT. Swirl vigorously; transfer the supernatants into a conical tube. Rinse the plate two times using RP10 medium and pool the supernatants into the collection tube.</li>
	<li>Centrifuge the collection tube at 400 x g, 4 &#176;C for 5 min. Aspirate the liquid and resuspend the pelleted cells in 5 ml RP10 medium. Add the cell suspension to a new panning plate, swirl and incubate for 30 min at RT. Collect the cells rinse the plate and pool them into a conical tube. Once TECs are enriched, spin down the cells at 400 x g, 4 &#176;C for 5 min and suspend cells in 5 ml FACS sorting buffer.</li>
</ol>
6. Purification of TECs by Cell Sorting
<ol>
	<li>Count cells using a hemocytometer and prepare cell suspension at a concentration of 4 x 107 cells in FACS sorting buffer. Add antibodies described in Section 4.4 into cell solution and incubate cells gently on a rotating mixer at 4 &#176;C for 30 min. Following staining, wash and suspend cells to a concentration of 10 x 106 cells per ml of FACS sorting buffer.</li>
	<li>Sort TEC subsets through a 100 &#181;M nozzle using fluorescence activated cell sorting. Sorted cell are collected in 30% (v/v) FBS in RPMI-1640). Once cell sorting is done, centrifuge cells at 400 x g, 4 &#176;C for 5 min and prepare for downstream analysis.</li>
</ol>

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The authors declare that they have no competing financial interests.

In the protocol, critical steps are the preparation of thymus stromal cells (Section 3) and the enrichment of TECs (Section 4). It is strongly recommended that fresh enzyme solution is prepared each time and the tissue is treated as soon as possible. For pooled thymi, optimizing the volume of enzyme solution is required depending upon the number of thymi. If any tissue residue is left after the processing, adding more enzyme solution and extension of incubation time could increase cell yield. During enrichment of TECs, c...

Early in T cell development, bone marrow hematopoietic stem cell-derived multipotent progenitors are recruited to the cortex of the thymus, undergo commitment to T lineage and become T cell precursors1. In the cortex, T cell precursor CD4 and CD8 double negative (DN) thymocytes expand and differentiate into immature CD4 and CD8 double positive (DP) thymocytes, forming a large pool of progenitors with highly variable T cell receptors1. Only a select MHC-restricted subset of DP cells will become CD4 or CD8 single positive (SP) thymocytes, migrate to the medulla of the thymus, and differentiate into functionally competent mature T cells, an event that is referred to as positive selection2-6. In contrast, clones of auto-reactive thymocytes undergo negative selection and are removed via apoptosis, converted to regulatory T cells for self-tolerance, or diverted to intraepithelial lymphocytes for purposes that are not yet clear3,7-10.
In the thymus, thymic stromal cells form a unique microenvironment providing signals for these various T cell development fates5,11,12. Thymic stromal cells are composed of thymic epithelial cells (TECs) - including cortical TECs (cTECs) and medullary TECs (mTECs), dendritic cells, macrophages, fibroblasts, endothelial cells, neural crest-derived pericytes and other mesenchymal cells13-15. Among these, TECs are crucial at the various stages of T cell development1,2,16,17. However, lack of a robust way to isolate TECs has hampered a comprehensive understanding of their functions16. In particular, cTECs, which form a three-dimensional network surrounding progenitors in the cortex, are essential for positive selection13,18,19 for reasons that are not yet clear. Earlier studies provided clues as to the heterogeneity and role of TECs, mostly relying on morphological and histological tools13. Recently the unique roles of TEC subsets were addressed by genetic approaches in mouse models12,20. A robust and reproducible way to isolate TECs is fundamental to achieve unbiased characterization of TEC subsets, quantitative and qualitative assessment of TEC functions, and clarification of mechanisms how cTECs support positive selection.
Due to the rarity of TECs in the thymus and the tight interactions they form in the intact organ, the isolation of TECs has been challenging. The protocol described here is based on previous discoveries, currently available reagents, techniques, and knowledge of thymus structure and stromal composition. Nearly two decades ago, several procedures were reported to disaggregate thymus tissues21-27, in which different enzymes were used during digestion, including Trypsin, Collagenase and Dispase. Gray et al. compared those enzymes in their precedure28, and reported an improved method with a multiple-step digestion of enzyme cocktails29 that became widely used20,30. However, this method involves a long preparation time and complex digestion steps and results in variable final cell numbers and proportions of TECs even in the same murine cohort29,30. Several years ago, Liberase research grade enzyme, containing highly purified Collagenase and neutral protease started to be used in thymus tissue dissociation30. Here, we describe a Liberase digestion-based protocol, with optimized mechanical separation procedures, that yields a high number of viable TECs from mouse thymus tissues.
To enrich dissociated stromal cells, previous studies have used either density gradients or magnetic bead separation21,29. However, both methods cause severe lost of certain populations of thymic stromal cells, particularly the population of cTECs29. Cytotoxic elimination and panning techniques31,32 have widely been used for depletion or separation of lymphocytes in the immunological field12,31. After comparison of these techniques, we established the current panning protocol for enrichment of TECs. The gentle condition during the enrichment procedure leads to less cell death and unbiased and increased TEC recovery.
The isolated thymus cell suspension described in Section 3 can be directly applied to flow cytometric analysis for identification and characterization of TEC subsets and dendritic cells. Section 4 describes a simple and useful way to identify TEC subsets using multiparameter flow cytometer. For experiments that seek to obtain purified cTECs or mTECs, TEC enrichment and cell sorting procedures can be found in Section 5 and 6.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:487px;">Anti-mouse EpCAM</td>
			<td style="width:153px;">eBioscience</td>
			<td style="width:133px;">XX-5791-YY*</td>
			<td style="width:163px;">Clone G8.8</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-mouse MHC II</td>
			<td>eBioscience</td>
			<td>XX-5321-YY*</td>
			<td>Clone M5/114.15.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-mouse Ly51</td>
			<td>eBioscience</td>
			<td>XX-5891-YY*</td>
			<td>Clone 6C3</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UEA-1</td>
			<td>Vector Laboratory</td>
			<td>FL-1061</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flow cytometer</td>
			<td>BD Biosciences&#160;</td>
			<td>LSRFortessa</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flow cell sorter</td>
			<td>BD Biosciences&#160;</td>
			<td>FACSAria</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FACS tubes</td>
			<td>BD Biosciences</td>
			<td>352052</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flow cytometry analysis software</td>
			<td>TreeStar - Flowjo</td>
			<td>FlowJo v7/9</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">*XX varies by fluorochrome and YY varies by vial size.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Using this protocol, a thymus organ was removed from an adult mouse (Section 2) and a thymic cell suspension was prepared as outlined in Section 3. The obtained cell suspension consisted of thymocytes, hematopoietic-derived stromal cells and non-hematopoietic stromal cells. CD45 is a hematopoietic pan-marker expressed on both thymocytes and hematopoietic stromal cells such as macrophages and dendritic cells. In contrast, TECs are not of hematopoietic origin and do not express CD4515. Cell staining and flow cyt...

Watch this Scientific Journal Video about Isolation, Identification, and Purification of Murine Thymic Epithelial Cells at JoVE.com

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

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 ...

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Research

JoVE Journal

Immunology and Infection

31.9K visualizações.  University of Minnesota.  Descrevemos aqui um método eficaz para o isolamento, identificação e purificação de células epiteliais do timo do rato (TEC). O protocolo pode ser utilizada para estudos da função do timo para o desenvolvimento normal das células T, disfunção do timo, e a reconstituição de células T. 

Vídeo: Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

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 ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

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 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 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 ...

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

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 ...

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.

This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Isolation and Identification of Extravascular Immune Cells of the Heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.