This research was supported by grants from the Israel Science Foundation (No. 1418/11) to AHH and (No. 1933/12) to AW, the German Israeli Foundation for young investigators (GIF Young) to AHH, and the Dr. I. Cabakoff Research Endowment Fund at the Hebrew University-Hadassah School of Dental Medicine to AHH and AW.

Prepare in advance:
<ul>
	<li>PBS + 2% FCS</li>
	<li>PBS + 2% FCS with 2 mg/ml of Collagenase Type II and 1 mg/ml of DNAse Type I (1 ml per sample)</li>
	<li>Sterilized surgical instruments</li>
	<li>0.5 M EDTA solution</li>
</ul>
1. Upper Gingival Excision Technique
<ol>
	<li>Euthanize mice using approved IACUC guidance.</li>
	<li>Cut both sides of the oral cavity including the cheeks and the mandible ramus with a sharp/blunt straight scissors.</li>
	<li>Pull down the mandible.</li>
	<li>Cut the soft and hard tissues in an imaginary line 1 mm behind the thirds molars with standard scissors. Direct the scissors perpendicular to the plane of the palatal bone (Figure 2A, blue lines).</li>
	<li>Incise the soft and hard tissues 2 mm behind the incisors (Figure 2A, blue lines).</li>
	<li>Pull down the anterior part of the mouth. The nasal cavity is observable after this step.</li>
	<li>Place the scissors parallel to the palate, put one blade into the nasal cavity, the other blade should incise at the vestibule. Cut until the posterior incision (Figure 2A, green lines). Repeat this step on both sides of the maxilla. At the end the maxilla is detached from the rest of the skull (Figure 2B).</li>
	<li>Remove the maxilla, cut it in the middle suture (Figure 2, black dashed line) and trim the palatal tissue of each hemi-maxilla until reaching the alveolar bone.</li>
	<li>Peel the gingival tissues (both hemi-maxillae) from their anterior border with Adson forceps (without teeth).</li>
	<li>Place the excised gingiva in a plate with PBS + 2% FCS.</li>
</ol>
2. Gingival Processing
<ol>
	<li>Put the excised gingiva in a tissue culture dish (35 x 10 mm) with 1 ml PBS + 2% FCS, 2 mg/ml of Collagenase Type II and 1 mg/ml of DNAse Type I.</li>
	<li>Mince well the tissue with an N&deg;15 sterile surgical blade.</li>
	<li>Transfer the tissue from the plate to a 15 ml conical test tube.</li>
	<li>Wash the tissue/cells remaining on the plate with 1 ml PBS + 2% FCS and transfer to the same test tube.</li>
	<li>Incubate in a shaker incubator for 20 min at 37 &deg;C, 200 rpm (recommendation: preheat the incubator).</li>
	<li>Add 20 &mu;l of EDTA 0.5 M and incubate for another 10 min at 37 &deg;C, 200 rpm.</li>
	<li>Fill the test tube up to 12 ml with PBS + 2% FCS and centrifuge at 4 &deg;C, 400 x g for 8 min (recommendation: pre-chill the centrifuge).</li>
	<li>Remove supernatant and resuspend cells with 2 ml PBS + 2% FCS.</li>
	<li>Filter the sample with a 70 &mu;m cell-strainer and save flow through.</li>
	<li>Centrifuge at 4 &deg;C, 320 x g for 5 min.</li>
	<li>Remove supernatant and resuspend cells with 300 &mu;l PBS + 2% FCS.</li>
	<li>Count the cells (approximately 5-10 x 105 cells are expected from a single maxilla).</li>
</ol>
3. Extracellular and Intracellular Antibody Staining for Flow Cytometry Analysis
It is recommended to work inside a hood, with lights off. It is critical to keep the cells in constant cold environment, as well as all the materials needed.
<ol>
	<li>Stain cells against extracellular molecules by adding 0.2-0.5 &mu;g of each selected antibody per 1 x 106 cells in a total volume of 100 &mu;l.</li>
	<li>Vortex briefly.</li>
	<li>Incubate the tubes for 15 min in the dark at 4 &deg;C.</li>
	<li>Wash cells with 2 ml PBS + 2% FCS.</li>
	<li>Centrifuge at 4 &deg;C, 320 x g for 5 min.</li>
	<li>Aspirate supernatant and resuspend cells by adding, drop wise, 500 &mu;l cold BD Cytoperm/Cytofix while vortexing.</li>
	<li>Incubate the tubes for 40 min at 4 &deg;C in the dark.</li>
	<li>Vortex briefly.</li>
	<li>Wash the cells twice with 1 ml cold BD Perm/Wash Buffer and spin at 800 x g for 7 min at 4 &deg;C.</li>
	<li>Aspirate supernatant except for 300 &mu;l.</li>
	<li>Stain cells intracellularly by adding 1 &mu;g of each selected antibody per 1 x 106 cells in a total volume of 100 &mu;l.</li>
	<li>Vortex briefly.</li>
	<li>Incubate for 30 min in the dark at 4 &deg;C.</li>
	<li>Wash the cells twice with 1 ml cold BD Perm/Wash Buffer and spin at 800 x g for 7 min at 4 &deg;C.</li>
	<li>Aspirate supernatant and resuspend cells by adding 300 &mu;l cold BD Perm/Wash Buffer.</li>
	<li>Vortex briefly.</li>
	<li>Transfer the suspension while filtering to FACS tubes.</li>
	<li>Keep the tubes in a dark and cold environment until analysis.</li>
</ol>

<ol>
	<li>Arizon, M., 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=Langerhans+cells+down-regulate+inflammation-driven+alveolar+bone+loss.">Langerhans cells down-regulate inflammation-driven alveolar bone loss.</a> Proc. Natl. Acad. Sci. U.S.A. 109, 7043-7048 (2012).</li><li>Listgarten, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+periodontitis.">Pathogenesis of periodontitis.</a> J. Clin. Periodontol. 13, 418-430 (1986).</li><li>Kinane, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causation+and+pathogenesis+of+periodontal+disease.">Causation and pathogenesis of periodontal disease.</a> Periodontol. 25, 8-20 (2000).</li><li>Jotwani, R., 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=Mature+dendritic+cells+infiltrate+the+T+cell-rich+region+of+oral+mucosa+in+chronic+periodontitis:+in+situ,+in+vivo,+and+in+vitro+studies.">Mature dendritic cells infiltrate the T cell-rich region of oral mucosa in chronic periodontitis: in situ, in vivo, and in vitro studies.</a> J. Immunol. 167, 4693-4700 (2001).</li><li>Graves, D. T., 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=Interleukin-1+and+tumor+necrosis+factor+antagonists+inhibit+the+progression+of+inflammatory+cell+infiltration+toward+alveolar+bone+in+experimental+periodontitis.">Interleukin-1 and tumor necrosis factor antagonists inhibit the progression of inflammatory cell infiltration toward alveolar bone in experimental periodontitis.</a> J. Periodontol. 69, 1419-1425 (1998).</li><li>Kabashima, H., Yoneda, M., Nagata, K., Hirofuji, T., Maeda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+presence+of+chemokine+(MCP-1,+MIP-1alpha,+MIP-1beta,+IP-10,+RANTES)-positive+cells+and+chemokine+receptor+(CCR5,+CXCR3)-positive+cells+in+inflamed+human+gingival+tissues.">The presence of chemokine (MCP-1, MIP-1alpha, MIP-1beta, IP-10, RANTES)-positive cells and chemokine receptor (CCR5, CXCR3)-positive cells in inflamed human gingival tissues.</a> Cytokine. 20, 70-77 (2002).</li><li>Moughal, N. A., Adonogianaki, E., Kinane, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Langerhans+cell+dynamics+in+human+gingiva+during+experimentally+induced+inflammation.">Langerhans cell dynamics in human gingiva during experimentally induced inflammation.</a> J. Biol. Buccale. 20, 163-167 (1992).</li><li>Cochran, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+bone+loss+in+periodontal+disease.">Inflammation and bone loss in periodontal disease.</a> J. Periodontol. 79, 1569-1576 (2008).</li><li>Taubman, M. A., Valverde, P., Han, X., Kawai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+response:+the+key+to+bone+resorption+in+periodontal+disease.">Immune response: the key to bone resorption in periodontal disease.</a> J. Periodontol. 76, 2033-2041 (2005).</li><li>Graves, D. T., Kang, J., Andriankaja, O., Wada, K., Rossa, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+to+study+host-bacteria+interactions+involved+in+periodontitis.">Animal models to study host-bacteria interactions involved in periodontitis.</a> Front Oral Biol. 15, 117-132 (2012).</li></ol>

Authors have nothing to disclose.

Maxilla gingival tissues obtained from a single mouse are sufficient for analyzing sub-populations of T and B lymphocytes, as well as their capability to express extracellular and intracellular molecules as we previously described 1. Nevertheless, if rare cell populations are of interest (for example DCs), it is recommended to pool tissues from 2-3 mice. Of note, if preferable, it is possible to peel both the palatal and gingival tissues and then to excise the gingiva (a modification of step 1.8-1.9). It shoul...

Gingiva is the soft tissue surrounding the cervical portion of the teeth and covers the alveolar process (Figure 1). The gingiva is a type of masticatory mucosa that can be further separated into mucosal epithelium and connective tissue (also known as submucosa or lamina propria). The anatomical structure of the gingiva and adjacent teeth allows bacteria to reside and develop plaque (biofilm) that constantly challenges the local immune system. As a result, inflammatory response develops in the gingiva, which in certain circumstances becomes destructive - a condition termed periodontal diseases 2. Basically, plaque-induced periodontal pathologies can be divided into gingivitis and periodontitis. Gingivitis represents a reversible condition of local inflammatory response that is confined to the gingiva. Periodontitis, on the other hand, is an irreversible destructive process in which the attachment apparatus (alveolar bone, periodontal ligament, cementum and gingiva) is destroyed 3.
The gingiva was proposed to serve both as effector and inductive sites during periodontal diseases 4. Human studies have suggested that in response to dental plaque, immune effector cells and molecules dynamically infiltrated or departure the gingiva 5-7. This activity was shown to play a major role in periodontal destruction 8,9. Whereas data generated by those studies provided valuable information regarding this pathological process, working with human tissues possess major ethical, technical and experimental limitations. The development of experimental models allowed cause-effect experimentations via employing transgenic mice and in vivo interventions 10. As a result, our knowledge on the mechanisms involved in periodontal disease increased considerably during the last two decades. Even so, due to the complexity of periodontal diseases, there is an ongoing debate regarding the nature of local immune response facilitating tissue destruction. There is also a lack in our understanding on the function of central immune cells in the gingiva during periodontal diseases. It is thus essential to study pathological inflammatory events occurring in the target tissue of the disease, the gingiva.

<table border="1">
 <tbody>
 <tr>
 <td>Comments (optional)</td>
 <td>Catalogue number</td>
 <td>Company</td>
 <td>Name of the reagent</td>
 </tr>
 <tr>
 <td></td>
 <td>CLS-2</td>
 <td>Worthington Biochemical Corp.</td>
 <td>Collagenase Type II</td>
 </tr>
 <tr>
 <td></td>
 <td>DN25-1G</td>
 <td>SIGMA</td>
 <td>DNAse I</td>
 </tr>
 <tr>
 <td></td>
 <td>E6758-100G</td>
 <td>SIGMA</td>
 <td>EDTA</td>
 </tr>
 <tr>
 <td></td>
 <td>D8537</td>
 <td>SIGMA</td>
 <td>Dulbecco's PBS</td>
 </tr>
 <tr>
 <td>Heat Inactivated</td>
 <td>04-121-1</td>
 <td>Biological Industries</td>
 <td>Fetal Bovine Serum </td>
 </tr>
 <tr>
 <td></td>
 <td>FPE-204-500</td>
 <td>Jet Biofil</td>
 <td>Vacuum-Driven Filter</td>
 </tr>
 <tr>
 <td></td>
 <td>352052</td>
 <td>BD Falcon</td>
 <td>5 ml Polystyrene Round-Bottom Tube</td>
 </tr>
 <tr>
 <td></td>
 <td>93070</td>
 <td>SPL Lifesciences</td>
 <td>Cell Strainer 70 &mu;m</td>
 </tr>
 <tr>
 <td></td>
 <td>153066</td>
 <td>NUNC</td>
 <td>Tissue Culture Dish 35&times;10 mm</td>
 </tr>
 <tr>
 <td></td>
 <td>554714</td>
 <td>BD</td>
 <td>BD Cytofix/Cytoperm</td>
 </tr>
 <tr>
 <td>Clone N418</td>
 <td>117305</td>
 <td>Biolegend</td>
 <td>Anti-mouse CD11c antibody</td>
 </tr>
 <tr>
 <td>Clone 104</td>
 <td>109819</td>
 <td>Biolegend</td>
 <td>Anti-mouse CD45.2 antibody</td>
 </tr>
 <tr>
 <td>Clone GK1.5</td>
 <td>100413</td>
 <td>Biolegend</td>
 <td>Anti-mouse CD4 antibody</td>
 </tr>
 <tr>
 <td>Clone 53-6.7</td>
 <td>100733</td>
 <td>Biolegend</td>
 <td>Anti-mouse CD8a antibody</td>
 </tr>
 <tr>
 <td>Clone 17A2</td>
 <td>100214</td>
 <td>Biolegend</td>
 <td>Anti-mouse CD3 antibody</td>
 </tr>
 <tr>
 <td>Clone G8.8</td>
 <td>118219</td>
 <td>Biolegend</td>
 <td>Anti-mouse CD326 (Ep-CAM) antibody</td>
 </tr>
 <tr>
 <td>Clone 929F3.01</td>
 <td>DDX0362D</td>
 <td>Imgenex</td>
 <td>Anti-mouse CD207 (Langerin) antibody</td>
 </tr>
 <tr>
 <td>Clone 39-10-8</td>
 <td>115010</td>
 <td>Biolegend</td>
 <td>Anti-mouse I-Ad (MHC-II) antobody</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td>BD Biosciences</td>
 <td>LSR II Flow Cytometer</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td>Tree Star</td>
 <td>FlowJo Software v 7.6.5</td>
 </tr>
 </tbody>
</table>

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.

Examples of flow cytometry analysis on gingival cells are presented. Gingival cells pooled from 2 mice were run in an LSR II flow cytometer and analyzed using FlowJo software. Figure 3A demonstrated the distribution of gingival cells from na&iuml;ve mice in a side scatter (SSC) versus forward scatter (FSC) plot. Gating strategy to identify (i) lymphocytes (ii) monocytes / dendritic cells and (iii) granulocytes is indicated. For comparison purpose, we also present a FACS plot illustrating gingival cells p...

Watch this Scientific Journal Video about Isolation, Processing and Analysis of Murine Gingival Cells at JoVE.com

Isolation, Processing and Analysis of Murine Gingival Cells

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 ...

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Immunology and Infection

18.8K Aufrufe.  Hebrew University - Hadassah Medical Center. Diese Studie beschreibt ein effizientes Verfahren zur Isolation und Behandlung Gingiva von der Maus Mundhöhle, um eine Einzelzellen-Kultur zu erzeugen. Die resultierenden Zellen können weiter für die Durchflusszytometrie und molekulare Studien eingesetzt werden.

Serie: Isolation, Processing and Analysis of Murine Gingival Cells

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

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 ...

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 of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

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 ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...